Anti-PD-L1 antibodies and uses thereof

ABSTRACT

Provided are anti-PD-L1 antibodies or fragments thereof. The antibodies or fragments thereof specifically bind to the immunoglobulin C domain of the PD-L1 protein. In various example, the antibodies or fragments thereof include a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6, or variants of each thereof. Methods of using the antibodies or fragments thereof for treating and diagnosing diseases such as cancer and infectious diseases are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is the U.S. national stage application ofInternational Application PCT/CN2017/088033, filed Jun. 13, 2017, whichclaims priority to International Application PCT/CN2017/072566, filedJan. 25, 2017 and Chinese Patent Application No. 201610414226.5, filedJun. 13, 2016, the contents of which are incorporated herein byreference in their entireties in the present disclosure.

BACKGROUND

Programmed death-ligand 1 (PD-L1), also known as cluster ofdifferentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a 40 kDa type 1transmembrane protein believed to play a major role in suppressing theimmune system during particular events such as pregnancy, tissueallografts, autoimmune disease and other disease states such ashepatitis. The binding of PD-L1 to PD-1 or B7.1 transmits an inhibitorysignal which reduces the proliferation of CD8+ T cells at the lymphnodes and supplementary to that PD-1 is also able to control theaccumulation of foreign antigen specific T cells in the lymph nodesthrough apoptosis which is further mediated by a lower regulation of thegene Bcl-2.

It has been shown that upregulation of PD-L1 may allow cancers to evadethe host immune system. An analysis of tumor specimens from patientswith renal cell carcinoma found that high tumor expression of PD-L1 wasassociated with increased tumor aggressiveness and an increased risk ofdeath. Many PD-L1 inhibitors are in development as immuno-oncologytherapies and are showing good results in clinical trials.

In addition to treatment of cancers, PD-L1 inhibition has also shownpromises in treating infectious diseases. In a mouse model ofintracellular infection, L. monocytogenes induced PD-L1 proteinexpression in T cells, NK cells, and macrophages. PD-L1 blockade (e.g.,using blocking antibodies) resulted in increased mortality for infectedmice. Blockade reduced TNFα and nitric oxide production by macrophages,reduced granzyme B production by NK cells, and decreased proliferationof L. monocytogenes antigen-specific CD8 T cells (but not CD4 T cells).This evidence suggests that PD-L1 acts as a positive costimulatorymolecule in intracellular infection.

SUMMARY

The present disclosure provides anti-PD-L1 antibody having high bindingaffinity to human PD-L1 proteins and can effectively block theinteraction between PD-L1 and its receptor PD-1. Also importantly, theexamples demonstrate that these anti-PD-L1 antibodies promote T cellimmune response and inhibit tumor growth. Different from knownanti-PD-L1 antibodies that bind to the immunoglobulin V domain of theextracellular portion of the PD-L1 protein, these antibodies bind to theimmunoglobulin C domain, in particular amino acid residues Y134, K162,and N183. These anti-PD-L1 antibodies are useful for therapeuticpurposes such as treating various types of cancer, as well asinfections, and can also be used for diagnostic and prognostic purposes.

One embodiment of the present disclosure provides an anti-PD-L1 antibodyor fragment thereof, which antibody or fragment thereof can specificallybind to an immunoglobulin C (Ig C) domain of a human Programmeddeath-ligand 1 (PD-L1) protein. In some embodiments, the Ig C domainconsists of amino acid residues 133-225. In some embodiments, theantibody or fragment thereof can bind to at least one of amino acidresidues Y134, K162, or N183 of the PD-L1 protein. In some embodiments,the antibody or fragment thereof can bind to at least one of amino acidresidues Y134, K162, and N183 of the PD-L1 protein. In some embodiments,the antibody or fragment thereof does not bind to an immunoglobulin V(Ig V) domain of the PD-L1 protein, wherein the Ig V domain consists ofamino acid residues 19-127.

One embodiment of the present disclosure provides an anti-PD-L1 antibodyor fragment thereof, wherein the antibody or fragment thereof hasspecificity to a human Programmed death-ligand 1 (PD-L1) protein andcomprises a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VHCDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO:5, and a VL CDR3 of SEQ ID NO: 6. In some embodiments, the antibody orfragment thereof further comprises a heavy chain constant region, alight chain constant region, an Fc region, or the combination thereof.In some embodiments, the light chain constant region is a kappa orlambda chain constant region. In some embodiments, the antibody orfragment thereof is of an isotype of IgG, IgM, IgA, IgE or IgD. In someembodiments, the isotype is IgG1, IgG2, IgG3 or IgG4. Withoutlimitation, the antibody or fragment thereof is a chimeric antibody, ahumanized antibody, or a fully human antibody. In one aspect, antibodyor fragment thereof is a humanized antibody.

In some embodiments, the antibody or fragment thereof comprises a heavychain variable region comprising one or more amino acid residuesselected from the group consisting of (a) Ser at position 44, (b) Ala atposition 49, (c) Ala at position 53, (d) Ile at position 91, (e) Glu atposition 1, (f) Val at position 37, (g) Thr at position 40 (h) Val atposition 53, (i) Glu at position 54, (j) Asn at position 77, (k) Arg atposition 94, and (l) Thr at position 108, according to Kabat numbering,and combinations thereof. In some embodiments, the antibody or fragmentthereof comprises a heavy chain variable region comprising (a) Ser atposition 44, (b) Ala at position 49, (c) Ala at position 53, and/or (d)Ile at position 91, according to Kabat numbering, and combinationsthereof.

In some embodiments, the antibody or fragment thereof comprises a lightchain variable region comprising one or more amino acid residuesselected from the group consisting of: (a) Ser at position 22, (b) Glnat position 42, (c) Ser at position 43, (d) Asp at position 60, and (e)Thr at position 63, according to Kabat numbering, and combinationsthereof.

Non-limiting examples of antibody or fragment thereof include thosehaving a heavy chain variable region comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 7-26, or a peptidehaving at least 90% sequence identity to an amino acid sequence selectedfrom the group consisting of SEQ ID NO: 7-26. Non-limiting examples ofantibody or fragment thereof include those having a light chain variableregion comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 27-33, or a peptide having at least 90%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NO: 27-33. Non-limiting examples of antibody orfragment thereof include those having a heavy chain variable regioncomprising the amino acid sequence of SEQ ID NO: 20 and a light chainvariable region comprising the amino acid sequence of SEQ ID NO: 28.

Biologically equivalent variants of the antibodies and fragments thereofare also described. In some embodiments, provided is an isolatedantibody or fragment thereof, wherein the antibody or fragment thereofhas specificity to a human PD-L1 protein and comprises: (a) a VH CDR1 ofSEQ ID NO: 1, or a variant of SEQ ID NO: 1 having a single substitution,deletion or insertion at location 1, 2 or 5 of SEQ ID NO: 1; (b) a VHCDR2 of SEQ ID NO: 2, or a variant of SEQ ID NO: 2 having a singlesubstitution, deletion or insertion at location 7, 8, 14 or 15 of SEQ IDNO: 2; (c) a VH CDR3 of SEQ ID NO: 3, or a variant of SEQ ID NO: 3having a single substitution, deletion or insertion at location 1, 2, 3,4, 5 or 6 of SEQ ID NO: 3; (d) a VL CDR1 of SEQ ID NO: 4, or a variantof SEQ ID NO: 4 having a single substitution, deletion or insertion atlocation 3 of SEQ ID NO: 4; (e) a VL CDR2 of SEQ ID NO: 5, or a variantof SEQ ID NO: 5 having a single substitution, deletion or insertion atlocation 1, 2, 3, 4, 5 or 6 of SEQ ID NO: 5 and (f) a VL CDR3 of SEQ IDNO: 6, or a variant of SEQ ID NO: 6 having a single substitution,deletion or insertion at location 11 or 2 of SEQ ID NO: 6.

In some embodiments, the variant of SEQ ID NO: 1 is selected from thegroup consisting of SEQ ID NO: 61-67. In some embodiments, the variantof SEQ ID NO: 2 is selected from the group consisting of SEQ ID NO:68-77. In some embodiments, the variant of SEQ ID NO: 3 is selected fromthe group consisting of SEQ ID NO: 78-90. In some embodiments, thevariant of SEQ ID NO: 4 is selected from the group consisting of SEQ IDNO: 91-92. In some embodiments, the variant of SEQ ID NO: 5 is selectedfrom the group consisting of SEQ ID NO: 93-105. In some embodiments, thevariant of SEQ ID NO: 6 is selected from the group consisting of SEQ IDNO: 106-111.

In some embodiments, the antibody or fragment thereof comprises a heavychain variable region comprising one or more amino acid residuesselected from the group consisting of (a) Ser at position 44, (b) Ala atposition 49, (c) Ala at position 53, (d) Ile at position 91, (e) Glu atposition 1, (f) Val at position 37, (g) Thr at position 40 (h) Val atposition 53, (i) Glu at position 54, (j) Asn at position 77, (k) Arg atposition 94, and (l) Thr at position 108, according to Kabat numbering,and combinations thereof. In some embodiments, the antibody or fragmentthereof comprises a heavy chain variable region comprising (a) Ser atposition 44, (b) Ala at position 49, (c) Ala at position 53, and/or (d)Ile at position 91, according to Kabat numbering, and combinationsthereof.

In some embodiments, the antibody or fragment thereof comprises a lightchain variable region comprising one or more amino acid residuesselected from the group consisting of: (a) Ser at position 22, (b) Glnat position 42, (c) Ser at position 43, (d) Asp at position 60, and (e)Thr at position 63, according to Kabat numbering, and combinationsthereof.

Also provided, in some embodiments, is a composition comprising theantibody or fragment thereof of the present disclosure and apharmaceutically acceptable carrier. Still also provided, in someembodiments, is an isolated cell comprising one or more polynucleotideencoding the antibody or fragment thereof of the present disclosure.

Treatment methods and uses are also provided. In one embodiment, amethod of treating cancer or infection in a patient in need thereof isprovided, comprising administering to the patient an effective amount ofthe antibody or fragment thereof of the present disclosure. In someembodiments, the cancer is a solid tumor. In some embodiments, thecancer is selected from the group consisting of bladder cancer, livercancer, colon cancer, rectal cancer, endometrial cancer, leukemia,lymphoma, pancreatic cancer, small cell lung cancer, non-small cell lungcancer, breast cancer, urethral cancer, head and neck cancer,gastrointestinal cancer, stomach cancer, oesophageal cancer, ovariancancer, renal cancer, melanoma, prostate cancer and thyroid cancer. Insome embodiments, the cancer is selected from the group consisting ofbladder cancer, liver cancer, pancreatic cancer, non-small cell lungcancer, breast cancer, urethral cancer, colorectal cancer, head and neckcancer, squamous cell cancer, Merkel cell carcinoma, gastrointestinalcancer, stomach cancer, oesophageal cancer, ovarian cancer, renalcancer, and small cell lung cancer. In some embodiments, the methodfurther comprises administering to the patient a second cancertherapeutic agent. In some embodiments, the infection is viralinfection, bacterial infection, fungal infection or infection by aparasite.

In another embodiment, a method of treating cancer or infection in apatient in need thereof is provided, comprising: (a) treating a cell, invitro, with the antibody or fragment thereof of the present disclosure;and (b) administering the treated cell to the patient. In someembodiments, the method further comprises, prior to step (a), isolatingthe cell from an individual. In some embodiments, the cell is isolatedfrom the patient. In some embodiments, the cell is isolated from a donorindividual different from the patient. In some embodiments, the cell isa T cell, non-limiting examples of which include a tumor-infiltrating Tlymphocyte, a CD4+ T cell, a CD8+ T cell, or the combination thereof.

Diagnostic methods and uses are also provided. In one embodiment, amethod of detecting expression of PD-L1 in a sample is provided,comprising contacting the sample with an antibody or fragment thereofunder conditions for the antibody or fragment thereof to bind to thePD-L1, and detecting the binding which indicates expression of PD-L1 inthe sample. In some embodiments, the sample comprises a tumor cell, atumor tissue, an infected tissue, or a blood sample.

Antibodies and fragment of the present disclosure can be used to preparebispecific antibodies. In one embodiment, an isolated bispecificantibody is provided, comprising a fragment of the present disclosureand a second antigen-binding fragment having specificity to a molecularon an immune cell. In some embodiments, the molecule is selected fromthe group consisting of PD-1, CTLA-4, LAG-3, CD28, CD122, 4-1BB, TIM3,OX-40, OX40L, CD40, CD40L, LIGHT, ICOS, ICOSL, GITR, GITRL, TIGIT, CD27,VISTA, B7H3, B7H4, HEVM or BTLA, CD47 and CD73. In some embodiments, thefragment and the second fragment each is independently selected from aFab fragment, a single-chain variable fragment (scFv), or asingle-domain antibody. In some embodiments, the bispecific antibodyfurther comprises a Fc fragment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that HL1210-3 can bind to human PD-L1 with high affinity.

FIG. 2 shows that HL1210-3 can efficiently inhibit the binding of humanPD-L1 to human PD1.

FIG. 3 shows the HL1210-3 antibody can highly efficiently inhibit thebinding of PD-1 on PD-L1 expressed on mammalian cells.

FIG. 4 shows that the tested anti-PD-L1 antibodies can promote human Tcell response.

FIG. 5 shows the binding kinetics of HL1210-3 to recombinant PD-L1.

FIG. 6 shows that all tested humanized antibodies had comparable bindingefficacy to human PD-L1 in contact to chimeric antibody.

FIG. 7 shows that all tested humanized antibodies can high efficientlybind to PD-L1 expressed on mammalian cells, comparable with chimericantibody.

FIG. 8 shows that humanized antibody Hu1210-41 can bind to rhesus PD-L1with lower affinity and cannot bind to rat and mouse PD-L1.

FIG. 9 shows that Hu1210-41 antibody can only specifically binding toB7-H1 (PD-L1), not B7-DC, B7-1, B7-2, B7-H2, PD-1, CD28, CTLA4, ICOS andBTLA.

FIG. 10 shows that Hu1210-41 can efficiently inhibit the binding ofhuman PD-L1 to human PD1 and B7-1.

FIG. 11 shows that Hu1210-41 can efficiently inhibit the binding ofhuman PD-L1 to human PD1 and B7-1.

FIG. 12 shows that the Hu1210-8, Hu1210-9, Hu1210-16, Hu1210-17,Hu1210-21 and Hu1210-36 humanized antibodies can dose dependentlypromote the IFNγ and IL-2 production in mix lymphocyte reaction.

FIG. 13 shows that the Hu1210-40, Hu1210-41 and Hu1210-17 humanizedantibodies can dose dependently promote the IFNγ production in CMVrecall assay.

FIG. 14 shows that Hu1210-31 can inhibit the tumor growth by 30% at 5mg/kg in HCC827-NSG-xenograft model.

FIG. 15 shows that Hu1210-41 antibody can dose-dependently inhibit thetumor growth in HCC827-NSG-xenograft model, while the tumor weight wasalso dose-dependently suppressed by Hu1210-41 antibody.

FIG. 16 plots, for each PD-L1 mutant, the mean binding value as afunction of expression (control anti-PD-L1 mAb reactivity).

FIG. 17 illustrates the locations of Y134, K162, and N183, the residues(spheres) involved in binding to the anti-PD-L1 Hu1210-41 antibody.

DETAILED DESCRIPTION Definitions

It is to be noted that the term “a” or “an” entity refers to one or moreof that entity; for example, “an antibody,” is understood to representone or more antibodies. As such, the terms “a” (or “an”), “one or more,”and “at least one” can be used interchangeably herein.

As used herein, the term “polypeptide” is intended to encompass asingular “polypeptide” as well as plural “polypeptides,” and refers to amolecule composed of monomers (amino acids) linearly linked by amidebonds (also known as peptide bonds). The term “polypeptide” refers toany chain or chains of two or more amino acids, and does not refer to aspecific length of the product. Thus, peptides, dipeptides, tripeptides,oligopeptides, “protein,” “amino acid chain,” or any other term used torefer to a chain or chains of two or more amino acids, are includedwithin the definition of “polypeptide,” and the term “polypeptide” maybe used instead of, or interchangeably with any of these terms. The term“polypeptide” is also intended to refer to the products ofpost-expression modifications of the polypeptide, including withoutlimitation glycosylation, acetylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, or modification by non-naturally occurring amino acids. Apolypeptide may be derived from a natural biological source or producedby recombinant technology, but is not necessarily translated from adesignated nucleic acid sequence. It may be generated in any manner,including by chemical synthesis.

The term “isolated” as used herein with respect to cells, nucleic acids,such as DNA or RNA, refers to molecules separated from other DNAs orRNAs, respectively, that are present in the natural source of themacromolecule. The term “isolated” as used herein also refers to anucleic acid or peptide that is substantially free of cellular material,viral material, or culture medium when produced by recombinant DNAtechniques, or chemical precursors or other chemicals when chemicallysynthesized. Moreover, an “isolated nucleic acid” is meant to includenucleic acid fragments which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to cells or polypeptides which are isolated fromother cellular proteins or tissues. Isolated polypeptides is meant toencompass both purified and recombinant polypeptides.

As used herein, the term “recombinant” as it pertains to polypeptides orpolynucleotides intends a form of the polypeptide or polynucleotide thatdoes not exist naturally, a non-limiting example of which can be createdby combining polynucleotides or polypeptides that would not normallyoccur together.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology canbe determined by comparing a position in each sequence which may bealigned for purposes of comparison. When a position in the comparedsequence is occupied by the same base or amino acid, then the moleculesare homologous at that position. A degree of homology between sequencesis a function of the number of matching or homologous positions sharedby the sequences. An “unrelated” or “non-homologous” sequence sharesless than 40% identity, though preferably less than 25% identity, withone of the sequences of the present disclosure.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” toanother sequence means that, when aligned, that percentage of bases (oramino acids) are the same in comparing the two sequences. This alignmentand the percent homology or sequence identity can be determined usingsoftware programs known in the art, for example those described inAusubel et al. eds. (2007) Current Protocols in Molecular Biology.Preferably, default parameters are used for alignment. One alignmentprogram is BLAST, using default parameters. In particular, programs areBLASTN and BLASTP, using the following default parameters: Geneticcode=standard; filter=none; strand=both; cutoff=60; expect=10;Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE;Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Biologically equivalentpolynucleotides are those having the above-noted specified percenthomology and encoding a polypeptide having the same or similarbiological activity.

The term “an equivalent nucleic acid or polynucleotide” refers to anucleic acid having a nucleotide sequence having a certain degree ofhomology, or sequence identity, with the nucleotide sequence of thenucleic acid or complement thereof. A homolog of a double strandednucleic acid is intended to include nucleic acids having a nucleotidesequence which has a certain degree of homology with or with thecomplement thereof. In one aspect, homologs of nucleic acids are capableof hybridizing to the nucleic acid or complement thereof. Likewise, “anequivalent polypeptide” refers to a polypeptide having a certain degreeof homology, or sequence identity, with the amino acid sequence of areference polypeptide. In some aspects, the sequence identity is atleast about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%. In some aspects,the equivalent polypeptide or polynucleotide has one, two, three, fouror five addition, deletion, substitution and their combinations thereofas compared to the reference polypeptide or polynucleotide. In someaspects, the equivalent sequence retains the activity (e.g.,epitope-binding) or structure (e.g., salt-bridge) of the referencesequence.

Hybridization reactions can be performed under conditions of different“stringency”. In general, a low stringency hybridization reaction iscarried out at about 40° C. in about 10×SSC or a solution of equivalentionic strength/temperature. A moderate stringency hybridization istypically performed at about 50° C. in about 6×SSC, and a highstringency hybridization reaction is generally performed at about 60° C.in about 1×SSC. Hybridization reactions can also be performed under“physiological conditions” which is well known to one of skill in theart. A non-limiting example of a physiological condition is thetemperature, ionic strength, pH and concentration of Mg²⁺ normally foundin a cell.

A polynucleotide is composed of a specific sequence of four nucleotidebases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil(U) for thymine when the polynucleotide is RNA. Thus, the term“polynucleotide sequence” is the alphabetical representation of apolynucleotide molecule. This alphabetical representation can be inputinto databases in a computer having a central processing unit and usedfor bioinformatics applications such as functional genomics and homologysearching. The term “polymorphism” refers to the coexistence of morethan one form of a gene or portion thereof. A portion of a gene of whichthere are at least two different forms, i.e., two different nucleotidesequences, is referred to as a “polymorphic region of a gene”. Apolymorphic region can be a single nucleotide, the identity of whichdiffers in different alleles.

The terms “polynucleotide” and “oligonucleotide” are usedinterchangeably and refer to a polymeric form of nucleotides of anylength, either deoxyribonucleotides or ribonucleotides or analogsthereof. Polynucleotides can have any three-dimensional structure andmay perform any function, known or unknown. The following arenon-limiting examples of polynucleotides: a gene or gene fragment (forexample, a probe, primer, EST or SAGE tag), exons, introns, messengerRNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA,miRNA, recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes and primers. A polynucleotide can comprise modifiednucleotides, such as methylated nucleotides and nucleotide analogs. Ifpresent, modifications to the nucleotide structure can be impartedbefore or after assembly of the polynucleotide. The sequence ofnucleotides can be interrupted by non-nucleotide components. Apolynucleotide can be further modified after polymerization, such as byconjugation with a labeling component. The term also refers to bothdouble- and single-stranded molecules. Unless otherwise specified orrequired, any embodiment of this disclosure that is a polynucleotideencompasses both the double-stranded form and each of two complementarysingle-stranded forms known or predicted to make up the double-strandedform.

The term “encode” as it is applied to polynucleotides refers to apolynucleotide which is said to “encode” a polypeptide if, in its nativestate or when manipulated by methods well known to those skilled in theart, it can be transcribed and/or translated to produce the mRNA for thepolypeptide and/or a fragment thereof. The antisense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom.

As used herein, an “antibody” or “antigen-binding polypeptide” refers toa polypeptide or a polypeptide complex that specifically recognizes andbinds to an antigen. An antibody can be a whole antibody and any antigenbinding fragment or a single chain thereof. Thus the term “antibody”includes any protein or peptide containing molecule that comprises atleast a portion of an immunoglobulin molecule having biological activityof binding to the antigen. Examples of such include, but are not limitedto a complementarity determining region (CDR) of a heavy or light chainor a ligand binding portion thereof, a heavy chain or light chainvariable region, a heavy chain or light chain constant region, aframework (FR) region, or any portion thereof, or at least one portionof a binding protein.

The terms “antibody fragment” or “antigen-binding fragment”, as usedherein, is a portion of an antibody such as F(ab′)₂, F(ab)₂, Fab′, Fab,Fv, scFv and the like. Regardless of structure, an antibody fragmentbinds with the same antigen that is recognized by the intact antibody.The term “antibody fragment” includes aptamers, spiegelmers, anddiabodies. The term “antibody fragment” also includes any synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex.

A “single-chain variable fragment” or “scFv” refers to a fusion proteinof the variable regions of the heavy (V_(H)) and light chains (V_(L)) ofimmunoglobulins. In some aspects, the regions are connected with a shortlinker peptide of ten to about 25 amino acids. The linker can be rich inglycine for flexibility, as well as serine or threonine for solubility,and can either connect the N-terminus of the V_(H) with the C-terminusof the V_(L), or vice versa. This protein retains the specificity of theoriginal immunoglobulin, despite removal of the constant regions and theintroduction of the linker. ScFv molecules are known in the art and aredescribed, e.g., in U.S. Pat. No. 5,892,019.

The term antibody encompasses various broad classes of polypeptides thatcan be distinguished biochemically. Those skilled in the art willappreciate that heavy chains are classified as gamma, mu, alpha, delta,or epsilon (γ, μ, α, δ, ε) with some subclasses among them (e.g.,γ1-γ4). It is the nature of this chain that determines the “class” ofthe antibody as IgG, IgM, IgA IgG, or IgE, respectively. Theimmunoglobulin subclasses (isotypes) e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgG₅,etc. are well characterized and are known to confer functionalspecialization. Modified versions of each of these classes and isotypesare readily discernable to the skilled artisan in view of the instantdisclosure and, accordingly, are within the scope of the instantdisclosure. All immunoglobulin classes are clearly within the scope ofthe present disclosure, the following discussion will generally bedirected to the IgG class of immunoglobulin molecules. With regard toIgG, a standard immunoglobulin molecule comprises two identical lightchain polypeptides of molecular weight approximately 23,000 Daltons, andtwo identical heavy chain polypeptides of molecular weight53,000-70,000. The four chains are typically joined by disulfide bondsin a “Y” configuration wherein the light chains bracket the heavy chainsstarting at the mouth of the “Y” and continuing through the variableregion.

Antibodies, antigen-binding polypeptides, variants, or derivativesthereof of the disclosure include, but are not limited to, polyclonal,monoclonal, multispecific, human, humanized, primatized, or chimericantibodies, single chain antibodies, epitope-binding fragments, e.g.,Fab, Fab′ and F(ab′)₂, Fd, Fvs, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv), fragments comprising either aVK or VH domain, fragments produced by a Fab expression library, andanti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodiesto LIGHT antibodies disclosed herein). Immunoglobulin or antibodymolecules of the disclosure can be of any type (e.g., IgG, IgE, IgM,IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2)or subclass of immunoglobulin molecule.

Light chains are classified as either kappa or lambda (K, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VK) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CK) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen-binding site or amino-terminusof the antibody. The N-terminal portion is a variable region and at theC-terminal portion is a constant region; the CH3 and CK domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

As indicated above, the variable region allows the antibody toselectively recognize and specifically bind epitopes on antigens. Thatis, the VK domain and VH domain, or subset of the complementaritydetermining regions (CDRs), of an antibody combine to form the variableregion that defines a three dimensional antigen-binding site. Thisquaternary antibody structure forms the antigen-binding site present atthe end of each arm of the Y. More specifically, the antigen-bindingsite is defined by three CDRs on each of the VH and VK chains (i.e.CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3). In some instances,e.g., certain immunoglobulin molecules derived from camelid species orengineered based on camelid immunoglobulins, a complete immunoglobulinmolecule may consist of heavy chains only, with no light chains. See,e.g., Hamers-Casterman et al., Nature 363:446-448 (1993).

In naturally occurring antibodies, the six “complementarity determiningregions” or “CDRs” present in each antigen-binding domain are short,non-contiguous sequences of amino acids that are specifically positionedto form the antigen-binding domain as the antibody assumes its threedimensional configuration in an aqueous environment. The remainder ofthe amino acids in the antigen-binding domains, referred to as“framework” regions, show less inter-molecular variability. Theframework regions largely adopt a β-sheet conformation and the CDRs formloops which connect, and in some cases form part of, the β-sheetstructure. Thus, framework regions act to form a scaffold that providesfor positioning the CDRs in correct orientation by inter-chain,non-covalent interactions. The antigen-binding domain formed by thepositioned CDRs defines a surface complementary to the epitope on theimmunoreactive antigen. This complementary surface promotes thenon-covalent binding of the antibody to its cognate epitope. The aminoacids comprising the CDRs and the framework regions, respectively, canbe readily identified for any given heavy or light chain variable regionby one of ordinary skill in the art, since they have been preciselydefined (see “Sequences of Proteins of Immunological Interest,” Kabat,E., et al., U.S. Department of Health and Human Services, (1983); andChothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

In the case where there are two or more definitions of a term which isused and/or accepted within the art, the definition of the term as usedherein is intended to include all such meanings unless explicitly statedto the contrary. A specific example is the use of the term“complementarity determining region” (“CDR”) to describe thenon-contiguous antigen combining sites found within the variable regionof both heavy and light chain polypeptides. This particular region hasbeen described by Kabat et al., U.S. Dept. of Health and Human Services,“Sequences of Proteins of Immunological Interest” (1983) and by Chothiaet al., J. Mol. Biol. 196:901-917 (1987), which are incorporated hereinby reference in their entireties. The CDR definitions according to Kabatand Chothia include overlapping or subsets of amino acid residues whencompared against each other. Nevertheless, application of eitherdefinition to refer to a CDR of an antibody or variants thereof isintended to be within the scope of the term as defined and used herein.The appropriate amino acid residues which encompass the CDRs as definedby each of the above cited references are set forth in the table belowas a comparison. The exact residue numbers which encompass a particularCDR will vary depending on the sequence and size of the CDR. Thoseskilled in the art can routinely determine which residues comprise aparticular CDR given the variable region amino acid sequence of theantibody.

Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3  95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

Kabat et al. also defined a numbering system for variable domainsequences that is applicable to any antibody. One of ordinary skill inthe art can unambiguously assign this system of “Kabat numbering” to anyvariable domain sequence, without reliance on any experimental databeyond the sequence itself. As used herein, “Kabat numbering” refers tothe numbering system set forth by Kabat et al., U.S. Dept. of Health andHuman Services, “Sequence of Proteins of Immunological Interest” (1983).

In addition to table above, the Kabat number system describes the CDRregions as follows: CDR-H1 begins at approximately amino acid 31 (i.e.,approximately 9 residues after the first cysteine residue), includesapproximately 5-7 amino acids, and ends at the next tryptophan residue.CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includesapproximately 16-19 amino acids, and ends at the next arginine or lysineresidue. CDR-H3 begins at approximately the thirty third amino acidresidue after the end of CDR-H2; includes 3-25 amino acids; and ends atthe sequence W-G-X-G, where X is any amino acid. CDR-L1 begins atapproximately residue 24 (i.e., following a cysteine residue); includesapproximately 10-17 residues; and ends at the next tryptophan residue.CDR-L2 begins at approximately the sixteenth residue after the end ofCDR-L1 and includes approximately 7 residues. CDR-L3 begins atapproximately the thirty third residue after the end of CDR-L2 (i.e.,following a cysteine residue); includes approximately 7-11 residues andends at the sequence F or W-G-X-G, where X is any amino acid.

Antibodies disclosed herein may be from any animal origin includingbirds and mammals. Preferably, the antibodies are human, murine, donkey,rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. Inanother embodiment, the variable region may be condricthoid in origin(e.g., from sharks).

As used herein, the term “heavy chain constant region” includes aminoacid sequences derived from an immunoglobulin heavy chain. A polypeptidecomprising a heavy chain constant region comprises at least one of: aCH1 domain, a hinge (e.g., upper, middle, and/or lower hinge region)domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof.For example, an antigen-binding polypeptide for use in the disclosuremay comprise a polypeptide chain comprising a CH1 domain; a polypeptidechain comprising a CH1 domain, at least a portion of a hinge domain, anda CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3domain; a polypeptide chain comprising a CH1 domain, at least a portionof a hinge domain, and a CH3 domain, or a polypeptide chain comprising aCH1 domain, at least a portion of a hinge domain, a CH2 domain, and aCH3 domain. In another embodiment, a polypeptide of the disclosurecomprises a polypeptide chain comprising a CH3 domain. Further, anantibody for use in the disclosure may lack at least a portion of a CH2domain (e.g., all or part of a CH2 domain). As set forth above, it willbe understood by one of ordinary skill in the art that the heavy chainconstant region may be modified such that they vary in amino acidsequence from the naturally occurring immunoglobulin molecule.

The heavy chain constant region of an antibody disclosed herein may bederived from different immunoglobulin molecules. For example, a heavychain constant region of a polypeptide may comprise a CH1 domain derivedfrom an IgG₁ molecule and a hinge region derived from an IgG₃ molecule.In another example, a heavy chain constant region can comprise a hingeregion derived, in part, from an IgG₁ molecule and, in part, from anIgG₃ molecule. In another example, a heavy chain portion can comprise achimeric hinge derived, in part, from an IgG₁ molecule and, in part,from an IgG₄ molecule.

As used herein, the term “light chain constant region” includes aminoacid sequences derived from antibody light chain. Preferably, the lightchain constant region comprises at least one of a constant kappa domainor constant lambda domain.

A “light chain-heavy chain pair” refers to the collection of a lightchain and heavy chain that can form a dimer through a disulfide bondbetween the CL domain of the light chain and the CH1 domain of the heavychain.

As previously indicated, the subunit structures and three dimensionalconfiguration of the constant regions of the various immunoglobulinclasses are well known. As used herein, the term “VH domain” includesthe amino terminal variable domain of an immunoglobulin heavy chain andthe term “CH1 domain” includes the first (most amino terminal) constantregion domain of an immunoglobulin heavy chain. The CH1 domain isadjacent to the VH domain and is amino terminal to the hinge region ofan immunoglobulin heavy chain molecule.

As used herein the term “CH2 domain” includes the portion of a heavychain molecule that extends, e.g., from about residue 244 to residue 360of an antibody using conventional numbering schemes (residues 244 to360, Kabat numbering system; and residues 231-340, EU numbering system;see Kabat et al., U.S. Dept. of Health and Human Services, “Sequences ofProteins of Immunological Interest” (1983). The CH2 domain is unique inthat it is not closely paired with another domain. Rather, two N-linkedbranched carbohydrate chains are interposed between the two CH2 domainsof an intact native IgG molecule. It is also well documented that theCH3 domain extends from the CH2 domain to the C-terminal of the IgGmolecule and comprises approximately 108 residues.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen-binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al., J. Immunol161:4083 (1998)).

As used herein the term “disulfide bond” includes the covalent bondformed between two sulfur atoms. The amino acid cysteine comprises athiol group that can form a disulfide bond or bridge with a second thiolgroup. In most naturally occurring IgG molecules, the CH1 and CK regionsare linked by a disulfide bond and the two heavy chains are linked bytwo disulfide bonds at positions corresponding to 239 and 242 using theKabat numbering system (position 226 or 229, EU numbering system).

As used herein, the term “chimeric antibody” will be held to mean anyantibody wherein the immunoreactive region or site is obtained orderived from a first species and the constant region (which may beintact, partial or modified in accordance with the instant disclosure)is obtained from a second species. In certain embodiments the targetbinding region or site will be from a non-human source (e.g. mouse orprimate) and the constant region is human.

As used herein, “percent humanization” is calculated by determining thenumber of framework amino acid differences (i.e., non-CDR difference)between the humanized domain and the germline domain, subtracting thatnumber from the total number of amino acids, and then dividing that bythe total number of amino acids and multiplying by 100.

By “specifically binds” or “has specificity to,” it is generally meantthat an antibody binds to an epitope via its antigen-binding domain, andthat the binding entails some complementarity between theantigen-binding domain and the epitope. According to this definition, anantibody is said to “specifically bind” to an epitope when it binds tothat epitope, via its antigen-binding domain more readily than it wouldbind to a random, unrelated epitope. The term “specificity” is usedherein to qualify the relative affinity by which a certain antibodybinds to a certain epitope. For example, antibody “A” may be deemed tohave a higher specificity for a given epitope than antibody “B,” orantibody “A” may be said to bind to epitope “C” with a higherspecificity than it has for related epitope “D.”

As used herein, the terms “treat” or “treatment” refer to boththerapeutic treatment and prophylactic or preventative measures, whereinthe object is to prevent or slow down (lessen) an undesiredphysiological change or disorder, such as the progression of cancer.Beneficial or desired clinical results include, but are not limited to,alleviation of symptoms, diminishment of extent of disease, stabilized(i.e., not worsening) state of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treatment” can also mean prolonging survival as comparedto expected survival if not receiving treatment. Those in need oftreatment include those already with the condition or disorder as wellas those prone to have the condition or disorder or those in which thecondition or disorder is to be prevented.

By “subject” or “individual” or “animal” or “patient” or “mammal,” ismeant any subject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo, sport, or pet animals such asdogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, andso on.

As used herein, phrases such as “to a patient in need of treatment” or“a subject in need of treatment” includes subjects, such as mammaliansubjects, that would benefit from administration of an antibody orcomposition of the present disclosure used, e.g., for detection, for adiagnostic procedure and/or for treatment.

Anti-PD-L1 Antibodies

The present disclosure provides anti-PD-L1 antibodies with high affinityto the human PD-L1 protein. The tested antibodies exhibited potentbinding and inhibitory activities and are useful for therapeutic anddiagnostics uses.

The PD-L1 protein is a 40 kDa type 1 transmembrane protein. Itsextracellular portion includes an N-terminal immunoglobulin V (IgV)domain (amino acids 19-127) and a C-terminal immunoglobulin C (IgC)domain (amino acids 133-225). PD-1 and PD-L1 interact through theconserved front and side of their IgV domains, as do the IgV domains ofantibodies and T cell receptors. Not surprisingly, the currentanti-PD-L1 antibodies all bind to the IgV domain which can disrupt thebinding between PD-1 and PD-L1. It is therefore a surprising andunexpected finding of the present disclosure that antibodies, such asmany disclosed herein, that bind to the IgC domain of the PD-L1 proteincan still effectively, and perhaps even more so, inhibit PD-L1, leadingto even further improved therapeutic effects.

One embodiment of the present disclosure, therefore, provides ananti-PD-L1 antibody or fragment thereof, which antibody or fragmentthereof can specifically bind to an immunoglobulin C (Ig C) domain of ahuman Programmed death-ligand 1 (PD-L1) protein. In some embodiments,the Ig C domain consists of amino acid residues 133-225.

In some embodiments, the antibody or fragment thereof can bind to atleast one of amino acid residues Y134, K162, or N183 of the PD-L1protein. In some embodiments, the antibody or fragment thereof can bindto at least two of amino acid residues Y134, K162, or N183 of the PD-L1protein. In some embodiments, the antibody or fragment thereof can bindto at least one of amino acid residues Y134, K162, and N183 of the PD-L1protein. In some embodiments, the antibody or fragment thereof does notbind to an immunoglobulin V (Ig V) domain of the PD-L1 protein, whereinthe Ig V domain consists of amino acid residues 19-127.

In accordance with one embodiment of the present disclosure, provided isan antibody that includes the heavy chain and light chain variabledomains with the CDR regions as defined in SEQ ID NO: 1-6.

TABLE 1  Sequences of the CDR regions Name Sequence SEQ ID NO: VH CDR1SY DM S 1 VH CDR2 TISDGG GY IYYSD SV KG 2 VH CDR3 EF GKRY ALDY  3VL CDR1 KA S QDVTPAVA 4 VL CDR2 S TSSRYT 5 VL CDR3 QQ HYTTPLT 6

As demonstrated in the experimental examples, the antibodies thatcontained these CDR regions, whether mouse, humanized or chimeric, hadpotent PD-L1 binding and inhibitory activities. Further computermodeling indicated that certain residues within the CDR can be modifiedto retain or improve the property of the antibodies. Such residues arereferred to as “hot spots” which are underlined in Table 1. In someembodiments, an anti-PD-L1 antibody of the present disclosure includesthe VH and VL CDR as listed in Table 1, with one, two or three furthermodifications. Such modifications can be addition, deletion orsubstation of amino acids.

In some embodiments, the modification is substitution at no more thanone hot spot position from each of the CDRs. In some embodiments, themodification is substitution at one, two or three such hot spotpositions. In one embodiment, the modification is substitution at one ofthe hot spot positions. Such substitutions, in some embodiments, areconservative substitutions.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a nonessential amino acidresidue in an immunoglobulin polypeptide is preferably replaced withanother amino acid residue from the same side chain family. In anotherembodiment, a string of amino acids can be replaced with a structurallysimilar string that differs in order and/or composition of side chainfamily members.

Non-limiting examples of conservative amino acid substitutions areprovided in the table below, where a similarity score of 0 or higherindicates conservative substitution between the two amino acids.

TABLE 2 Amino Acid Similarity Matrix C G P S A T D E N Q H K R V M I L FY W W −8 −7 −6 −2 −6 −5 −7 −7 −4 −5 −3 −3 2 −6 −4 −5 −2 0 0 17 Y 0 −5 −5−3 −3 −3 −4 −4 −2 −4 0 −4 −5 −2 −2 −1 −1 7 10 F −4 −5 −5 −3 −4 −3 −6 −5−4 −5 −2 −5 −4 −1 0 1 2 9 L −6 −4 −3 −3 −2 −2 −4 −3 −3 −2 −2 −3 −3 2 4 26 I −2 −3 −2 −1 −1 0 −2 −2 −2 −2 −2 −2 −2 4 2 5 M −5 −3 −2 −2 −1 −1 −3−2 0 −1 −2 0 0 2 6 V −2 −1 −1 −1 0 0 −2 −2 −2 −2 −2 −2 −2 4 R −4 −3 0 0−2 −1 −1 −1 0 1 2 3 6 K −5 −2 −1 0 −1 0 0 0 1 1 0 5 H −3 −2 0 −1 −1 −1 11 2 3 6 Q −5 −1 0 −1 0 −1 2 2 1 4 N −4 0 −1 1 0 0 2 1 2 E −5 0 −1 0 0 03 4 D −5 1 −1 0 0 0 4 T −2 0 0 1 1 3 A −2 1 1 1 2 S 0 1 1 1 P −3 −1 6 G−3 5 C 12

TABLE 3 Conservative Amino Acid Substitutions For Amino AcidSubstitution With Alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys ArginineD-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-GluGln, D-Gln Aspartic Acid D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-GlnCysteine D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser GlutamineD-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid D-Glu, D-Asp,Asp, Asn, D-Asn, Gln, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-AlaIsoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val,Met, D-Met, D-Ile, D-Leu, Ile Lysine D-Lys, Arg, D-Arg, Orn, D-OrnMethionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-ValPhenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-ProSerine D-Ser, Thr, D-Thr, allo-Thr, L-Cys, D-Cys Threonine D-Thr, Ser,D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His,D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Specific examples of CDRs with suitable substitutions are provided inSEQ ID NO: 61-111 of Example 11. In some embodiments, therefore, anantibody of the present disclosure includes a VH CDR1 of SEQ ID NO: 1 orany one of 61-67. In some embodiments, an antibody of the presentdisclosure includes a VH CDR2 of SEQ ID NO: 2 or any one of 68-77. Insome embodiments, an antibody of the present disclosure includes a VHCDR3 of SEQ ID NO: 1 or any one of 78-90. In some embodiments, anantibody of the present disclosure includes a VL CDR1 of SEQ ID NO: 4 orany one of 91-92. In some embodiments, an antibody of the presentdisclosure includes a VL CDR2 of SEQ ID NO: 5 or any one of 93-105. Insome embodiments, an antibody of the present disclosure includes a VLCDR3 of SEQ ID NO: 6 or any one of 106-110.

In some embodiments, an antibody or fragment thereof includes no morethan one, no more than two, or no more than three of the abovesubstitutions. In some embodiments, the antibody or fragment thereofincludes a VH CDR1 of SEQ ID NO: 1 or any one of SEQ ID NO: 61-67, a VHCDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO:4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2 or any one of SEQ ID NO:68-77, a VH CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3 orany one of SEQ ID NO: 78-90, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQID NO: 5, and a VL CDR3 of SEQ ID NO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, aVL CDR1 of SEQ ID NO: 4 or any one of SEQ ID NO: 91-92, a VL CDR2 of SEQID NO: 5, and a VL CDR3 of SEQ ID NO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, aVL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5 or any one of SEQ IDNO: 93-105, and a VL CDR3 of SEQ ID NO: 6.

In some embodiments, the antibody or fragment thereof includes a VH CDR1of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, aVL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQID NO: 6 or any one of SEQ ID NO: 106-111.

Non-limiting examples of VH are provided in SEQ ID NO: 7-26 and 113, outof which SEQ ID NO: 113 is the mouse VH, and SEQ ID NO: 7-26 arehumanized ones. Further, among the humanized VH, SEQ ID NO: 9-15, 17-21and 23-26 include one or more back-mutations to the mouse version.Likewise, non-limiting examples of VL (VK) are provided in SEQ ID NO:27-33. SEQ ID NO: 28 and 30 are the originally derived, CDR-grafted,humanized sequences as shown in the examples. SEQ ID NO: 29 and 31-33are humanized VL with back-mutations.

The back-mutations are shown to be useful for retaining certaincharacteristics of the anti-PD-L1 antibodies. Accordingly, in someembodiments, the anti-PD-L1 antibodies of the present disclosure, inparticular the human or humanized ones, include one or more of theback-mutations. In some embodiments, the VH back-mutation (i.e.,included amino acid at the specified position) is one or more selectedfrom (a) Ser at position 44, (b) Ala at position 49, (c) Ala at position53, (d) Ile at position 91, (e) Glu at position 1, (f) Val at position37, (g) Thr at position 40 (h) Val at position 53, (i) Glu at position54, (j) Asn at position 77, (k) Arg at position 94, and (l) Thr atposition 108, according to Kabat numbering, and combinations thereof. Insome embodiments, the back-mutations are selected from (a) Ser atposition 44, (b) Ala at position 49, (c) Ala at position 53, and/or (d)Ile at position 91, according to Kabat numbering, and combinationsthereof.

In some embodiments, the VL back-mutation is one or more selected from(a) Ser at position 22, (b) Gln at position 42, (c) Ser at position 43,(d) Asp at position 60, and (e) Thr at position 63, according to Kabatnumbering, and combinations thereof.

In some embodiments, the anti-PD-L1 antibody of the present disclosureincludes a VH of SEQ ID NO: 7-26, a VL of SEQ ID NO: 27-33, or theirrespective biological equivalents. A biological equivalent of a VH or VLis a sequence that includes the designated amino acids while having anoverall 80%, 85%, 90%, 95%, 98% or 99% sequence identity. A biologicalequivalent of SEQ ID NO: 20, for instance, can be a VH that has anoverall 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO:20 but retains the CDRs (SEQ ID NO: 1-6 or their variants), andoptionally retains one or more, or all of the back-mutations. In oneembodiment, the VH has the amino acid sequence of SEQ ID NO: 20 and theVL has the amino acid sequence of SEQ ID NO: 28.

It will also be understood by one of ordinary skill in the art thatantibodies as disclosed herein may be modified such that they vary inamino acid sequence from the naturally occurring binding polypeptidefrom which they were derived. For example, a polypeptide or amino acidsequence derived from a designated protein may be similar, e.g., have acertain percent identity to the starting sequence, e.g., it may be 60%,70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the startingsequence.

In certain embodiments, the antibody comprises an amino acid sequence orone or more moieties not normally associated with an antibody. Exemplarymodifications are described in more detail below. For example, anantibody of the disclosure may comprise a flexible linker sequence, ormay be modified to add a functional moiety (e.g., PEG, a drug, a toxin,or a label).

Antibodies, variants, or derivatives thereof of the disclosure includederivatives that are modified, i.e., by the covalent attachment of anytype of molecule to the antibody such that covalent attachment does notprevent the antibody from binding to the epitope. For example, but notby way of limitation, the antibodies can be modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the antibodies may contain one or more non-classicalamino acids.

In some embodiments, the antibodies may be conjugated to therapeuticagents, prodrugs, peptides, proteins, enzymes, viruses, lipids,biological response modifiers, pharmaceutical agents, or PEG.

The antibodies may be conjugated or fused to a therapeutic agent, whichmay include detectable labels such as radioactive labels, animmunomodulator, a hormone, an enzyme, an oligonucleotide, a photoactivetherapeutic or diagnostic agent, a cytotoxic agent, which may be a drugor a toxin, an ultrasound enhancing agent, a non-radioactive label, acombination thereof and other such agents known in the art.

The antibodies can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantigen-binding polypeptide is then determined by detecting the presenceof luminescence that arises during the course of a chemical reaction.Examples of particularly useful chemiluminescent labeling compounds areluminol, isoluminol, theromatic acridinium ester, imidazole, acridiniumsalt and oxalate ester.

The antibodies can also be detectably labeled using fluorescenceemitting metals such as ¹⁵²Eu, or others of the lanthanide series. Thesemetals can be attached to the antibody using such metal chelating groupsas diethylenetriaminepentacetic acid (DTPA) orethylenediaminetetraacetic acid (EDTA). Techniques for conjugatingvarious moieties to an antibody are well known, see, e.g., Arnon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies ForDrug Delivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.,(eds.), Marcel Dekker, Inc., pp. 623-53 (1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), Academic Press pp. 303-16 (1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. (52:119-58 (1982)).

Bi-Functional Molecules

PD-L1 is an immune checkpoint molecule and is also a tumor antigen. As atumor antigen targeting molecule, an antibody or antigen-bindingfragment specific to PD-L1 can be combined with a second antigen-bindingfragment specific to an immune cell to generate a bispecific antibody.

In some embodiments, the immune cell is selected from the groupconsisting of a T cell, a B cell, a monocyte, a macrophage, aneutrophil, a dendritic cell, a phagocyte, a natural killer cell, aneosinophil, a basophil, and a mast cell. Molecules on the immune cellwhich can be targeted include, for example, CD3, CD16, CD19, CD28, andCD64. Other examples include PD-1, CTLA-4, LAG-3 (also known as CD223),CD28, CD122, 4-1BB (also known as CD137), TIM3, OX-40 or OX40L, CD40 orCD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27, VISTA, B7H3, B7H4,HEVM or BTLA (also known as CD272), killer-cell immunoglobulin-likereceptors (KIRs), and CD47. Specific examples of bispecificity include,without limitation, PD-L1/PD-1, PD-L1/LAG3, PD-L1/TIGIT, and PD-L1/CD47.

As an immune checkpoint inhibitor, an antibody or antigen-bindingfragment specific to PD-L1 can be combined with a second antigen-bindingfragment specific to a tumor antigen to generate a bispecific antibody.A “tumor antigen” is an antigenic substance produced in tumor cells,i.e., it triggers an immune response in the host. Tumor antigens areuseful in identifying tumor cells and are potential candidates for usein cancer therapy. Normal proteins in the body are not antigenic.Certain proteins, however, are produced or overexpressed duringtumorigenesis and thus appear “foreign” to the body. This may includenormal proteins that are well sequestered from the immune system,proteins that are normally produced in extremely small quantities,proteins that are normally produced only in certain stages ofdevelopment, or proteins whose structure is modified due to mutation.

An abundance of tumor antigens are known in the art and new tumorantigens can be readily identified by screening. Non-limiting examplesof tumor antigens include EGFR, Her2, EpCAM, CD20, CD30, CD33, CD47,CD52, CD133, CD73, CEA, gpA33, Mucins, TAG-72, CIX, PSMA, folate-bindingprotein, GD2, GD3, GM2, VEGF, VEGFR, Integrin, αVβ3, α5β1, ERBB2, ERBB3,MET, IGF1R, EPHA3, TRAILR1, TRAILR2, RANKL, FAP and Tenascin.

In some aspects, the monovalent unit has specificity to a protein thatis overexpressed on a tumor cell as compared to a correspondingnon-tumor cell. A “corresponding non-tumor cell” as used here, refers toa non-tumor cell that is of the same cell type as the origin of thetumor cell. It is noted that such proteins are not necessarily differentfrom tumor antigens. Non-limiting examples include carcinoembryonicantigen (CEA), which is overexpressed in most colon, rectum, breast,lung, pancreas and gastrointestinal tract carcinomas; heregulinreceptors (HER-2, neu or c-erbB-2), which is frequently overexpressed inbreast, ovarian, colon, lung, prostate and cervical cancers; epidermalgrowth factor receptor (EGFR), which is highly expressed in a range ofsolid tumors including those of the breast, head and neck, non-smallcell lung and prostate; asialoglycoprotein receptor; transferrinreceptor; serpin enzyme complex receptor, which is expressed onhepatocytes; fibroblast growth factor receptor (FGFR), which isoverexpressed on pancreatic ductal adenocarcinoma cells; vascularendothelial growth factor receptor (VEGFR), for anti-angiogenesis genetherapy; folate receptor, which is selectively overexpressed in 90% ofnonmucinous ovarian carcinomas; cell surface glycocalyx; carbohydratereceptors; and polymeric immunoglobulin receptor, which is useful forgene delivery to respiratory epithelial cells and attractive fortreatment of lung diseases such as Cystic Fibrosis. Non-limitingexamples of bispecificity in this respect include PD-L1/EGFR,PD-L1/Her2, PD-L1/CD33, PD-L1/CD133, PD-L1/CEA and PD-L1/VEGF.

Different format of bispecific antibodies are also provided. In someembodiments, each of the anti-PD-L1 fragment and the second fragmenteach is independently selected from a Fab fragment, a single-chainvariable fragment (scFv), or a single-domain antibody. In someembodiments, the bispecific antibody further includes a Fc fragment.

Bifunctional molecules that include not just antibody or antigen bindingfragment are also provided. As a tumor antigen targeting molecule, anantibody or antigen-binding fragment specific to PD-L1, such as thosedescribed here, can be combined with an immune cytokine or ligandoptionally through a peptide linker. The linked immune cytokines orligands include, but not limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7,IL-10, IL-12, IL-13, IL-15, GM-CSF, TNF-α, CD40L, OX40L, CD27L, CD30L,4-1BBL, LIGHT and GITRL. Such bi-functional molecules can combine theimmune checkpoint blocking effect with tumor site local immunemodulation.

Polynucleotides Encoding the Antibodies and Methods of Preparing theAntibodies

The present disclosure also provides isolated polynucleotides or nucleicacid molecules (e.g., SEQ ID NO: 34-60, 112, and 114) encoding theantibodies, variants or derivatives thereof of the disclosure. Thepolynucleotides of the present disclosure may encode the entire heavyand light chain variable regions of the antigen-binding polypeptides,variants or derivatives thereof on the same polynucleotide molecule oron separate polynucleotide molecules. Additionally, the polynucleotidesof the present disclosure may encode portions of the heavy and lightchain variable regions of the antigen-binding polypeptides, variants orderivatives thereof on the same polynucleotide molecule or on separatepolynucleotide molecules.

Methods of making antibodies are well known in the art and describedherein. In certain embodiments, both the variable and constant regionsof the antigen-binding polypeptides of the present disclosure are fullyhuman. Fully human antibodies can be made using techniques described inthe art and as described herein. For example, fully human antibodiesagainst a specific antigen can be prepared by administering the antigento a transgenic animal which has been modified to produce suchantibodies in response to antigenic challenge, but whose endogenous locihave been disabled. Exemplary techniques that can be used to make suchantibodies are described in U.S. Pat. Nos. 6,150,584; 6,458,592;6,420,140 which are incorporated by reference in their entireties.

In certain embodiments, the prepared antibodies will not elicit adeleterious immune response in the animal to be treated, e.g., in ahuman. In one embodiment, antigen-binding polypeptides, variants, orderivatives thereof of the disclosure are modified to reduce theirimmunogenicity using art-recognized techniques. For example, antibodiescan be humanized, primatized, deimmunized, or chimeric antibodies can bemade. These types of antibodies are derived from a non-human antibody,typically a murine or primate antibody, that retains or substantiallyretains the antigen-binding properties of the parent antibody, but whichis less immunogenic in humans. This may be achieved by various methods,including (a) grafting the entire non-human variable domains onto humanconstant regions to generate chimeric antibodies; (b) grafting at leasta part of one or more of the non-human complementarity determiningregions (CDRs) into a human framework and constant regions with orwithout retention of critical framework residues; or (c) transplantingthe entire non-human variable domains, but “cloaking” them with ahuman-like section by replacement of surface residues. Such methods aredisclosed in Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855(1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen etal., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 25:489-498(1991); Padlan, Molec. Immun. 31:169-217 (1994), and U.S. Pat. Nos.5,585,089, 5,693,761, 5,693,762, and 6,190,370, all of which are herebyincorporated by reference in their entirety.

De-immunization can also be used to decrease the immunogenicity of anantibody. As used herein, the term “de-immunization” includes alterationof an antibody to modify T-cell epitopes (see, e.g., InternationalApplication Publication Nos.: WO/9852976 A1 and WO/0034317 A2). Forexample, variable heavy chain and variable light chain sequences fromthe starting antibody are analyzed and a human T-cell epitope “map” fromeach V region showing the location of epitopes in relation tocomplementarity-determining regions (CDRs) and other key residues withinthe sequence is created. Individual T-cell epitopes from the T-cellepitope map are analyzed in order to identify alternative amino acidsubstitutions with a low risk of altering activity of the finalantibody. A range of alternative variable heavy and variable lightsequences are designed comprising combinations of amino acidsubstitutions and these sequences are subsequently incorporated into arange of binding polypeptides. Typically, between 12 and 24 variantantibodies are generated and tested for binding and/or function.Complete heavy and light chain genes comprising modified variable andhuman constant regions are then cloned into expression vectors and thesubsequent plasmids introduced into cell lines for the production ofwhole antibody. The antibodies are then compared in appropriatebiochemical and biological assays, and the optimal variant isidentified.

The binding specificity of antigen-binding polypeptides of the presentdisclosure can be determined by in vitro assays such asimmunoprecipitation, radioimmunoassay (RIA) or enzyme-linkedimmunoabsorbent assay (ELISA).

Alternatively, techniques described for the production of single-chainunits (U.S. Pat. No. 4,694,778; Bird, Science 242:423-442 (1988); Hustonet al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988); and Ward et al.,Nature 334:544-554 (1989)) can be adapted to produce single-chain unitsof the present disclosure. Single-chain units are formed by linking theheavy and light chain fragments of the Fv region via an amino acidbridge, resulting in a single-chain fusion peptide. Techniques for theassembly of functional Fv fragments in E. coli may also be used (Skerraet al., Science 242: 1038-1041 (1988)).

Examples of techniques which can be used to produce single-chain Fvs(scFvs) and antibodies include those described in U.S. Pat. Nos.4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88(1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerraet al., Science 240:1038-1040 (1988). For some uses, including in vivouse of antibodies in humans and in vitro detection assays, it may bepreferable to use chimeric, humanized, or human antibodies. A chimericantibody is a molecule in which different portions of the antibody arederived from different animal species, such as antibodies having avariable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region. Methods for producing chimericantibodies are known in the art. See, e.g., Morrison, Science 229:1202(1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J.Immunol. Methods 125:191-202 (1989); U.S. Pat. Nos. 5,807,715;4,816,567; and 4,816,397, which are incorporated herein by reference intheir entireties.

Humanized antibodies are antibody molecules derived from a non-humanspecies antibody that bind the desired antigen having one or morecomplementarity determining regions (CDRs) from the non-human speciesand framework regions from a human immunoglobulin molecule. Often,framework residues in the human framework regions will be substitutedwith the corresponding residue from the CDR donor antibody to alter,preferably improve, antigen-binding. These framework substitutions areidentified by methods well known in the art, e.g., by modeling of theinteractions of the CDR and framework residues to identify frameworkresidues important for antigen-binding and sequence comparison toidentify unusual framework residues at particular positions. (See, e.g.,Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323(1988), which are incorporated herein by reference in their entireties.)Antibodies can be humanized using a variety of techniques known in theart including, for example, CDR-grafting (EP 239,400; PCT publication WO91/09967; U.S. Pat. Nos. 5,225,539; 5,530,101; and 5,585,089), veneeringor resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology28(4/5):489-498 (1991); Studnicka et al., Protein Engineering7(6):805-814 (1994); Roguska. et al., Proc. Natl. Sci. USA 91:969-973(1994)), and chain shuffling (U.S. Pat. No. 5,565,332, which isincorporated by reference in its entirety).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods usingantibody libraries derived from human immunoglobulin sequences. Seealso, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO96/33735, and WO 91/10741; each of which is incorporated herein byreference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a desired target polypeptide. Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B-celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see Lonberg andHuszar Int. Rev. Immunol. 73:65-93 (1995). For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 96/34096; WO 96/33735; U.S. Pat. Nos.5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806;5,814,318; and 5,939,598, which are incorporated by reference herein intheir entirety. In addition, companies such as Abgenix, Inc. (Freemont,Calif.) and GenPharm (San Jose, Calif.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies which recognize a selected epitope can alsobe generated using a technique referred to as “guided selection.” Inthis approach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/Technology 72:899-903(1988). See also, U.S. Pat. No. 5,565,332, which is incorporated byreference in its entirety.)

In another embodiment, DNA encoding desired monoclonal antibodies may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Theisolated and subcloned hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into prokaryotic or eukaryotic host cellssuch as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)cells or myeloma cells that do not otherwise produce immunoglobulins.More particularly, the isolated DNA (which may be synthetic as describedherein) may be used to clone constant and variable region sequences forthe manufacture antibodies as described in Newman et al., U.S. Pat. No.5,658,570, filed Jan. 25, 1995, which is incorporated by referenceherein. Essentially, this entails extraction of RNA from the selectedcells, conversion to cDNA, and amplification by PCR using Ig specificprimers. Suitable primers for this purpose are also described in U.S.Pat. No. 5,658,570. As will be discussed in more detail below,transformed cells expressing the desired antibody may be grown up inrelatively large quantities to provide clinical and commercial suppliesof the immunoglobulin.

Additionally, using routine recombinant DNA techniques, one or more ofthe CDRs of the antigen-binding polypeptides of the present disclosure,may be inserted within framework regions, e.g., into human frameworkregions to humanize a non-human antibody. The framework regions may benaturally occurring or consensus framework regions, and preferably humanframework regions (see, e.g., Chothia et al., J. Mol. Biol. 278:457-479(1998) for a listing of human framework regions). Preferably, thepolynucleotide generated by the combination of the framework regions andCDRs encodes an antibody that specifically binds to at least one epitopeof a desired polypeptide, e.g., LIGHT. Preferably, one or more aminoacid substitutions may be made within the framework regions, and,preferably, the amino acid substitutions improve binding of the antibodyto its antigen. Additionally, such methods may be used to make aminoacid substitutions or deletions of one or more variable region cysteineresidues participating in an intrachain disulfide bond to generateantibody molecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentdisclosure and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA:851-855 (1984);Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule, ofappropriate antigen specificity, together with genes from a humanantibody molecule of appropriate biological activity can be used. Asused herein, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine monoclonal antibody and a humanimmunoglobulin constant region.

Yet another highly efficient means for generating recombinant antibodiesis disclosed by Newman, Biotechnology 10: 1455-1460 (1992).Specifically, this technique results in the generation of primatizedantibodies that contain monkey variable domains and human constantsequences. This reference is incorporated by reference in its entiretyherein. Moreover, this technique is also described in commonly assignedU.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which isincorporated herein by reference.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in thedisclosure as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Additionally, standard techniques known to those of skill in the art canbe used to introduce mutations in the nucleotide sequence encoding anantibody of the present disclosure, including, but not limited to,site-directed mutagenesis and PCR-mediated mutagenesis which result inamino acid substitutions. Preferably, the variants (includingderivatives) encode less than 50 amino acid substitutions, less than 40amino acid substitutions, less than 30 amino acid substitutions, lessthan 25 amino acid substitutions, less than 20 amino acid substitutions,less than 15 amino acid substitutions, less than 10 amino acidsubstitutions, less than 5 amino acid substitutions, less than 4 aminoacid substitutions, less than 3 amino acid substitutions, or less than 2amino acid substitutions relative to the reference variable heavy chainregion, CDR-H1, CDR-H2, CDR-H3, variable light chain region, CDR-L1,CDR-L2, or CDR-L3. Alternatively, mutations can be introduced randomlyalong all or part of the coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for biologicalactivity to identify mutants that retain activity.

Cancer Treatment

As described herein, the antibodies, variants or derivatives of thepresent disclosure may be used in certain treatment and diagnosticmethods.

The present disclosure is further directed to antibody-based therapieswhich involve administering the antibodies of the disclosure to apatient such as an animal, a mammal, and a human for treating one ormore of the disorders or conditions described herein. Therapeuticcompounds of the disclosure include, but are not limited to, antibodiesof the disclosure (including variants and derivatives thereof asdescribed herein) and nucleic acids or polynucleotides encodingantibodies of the disclosure (including variants and derivatives thereofas described herein).

The antibodies of the disclosure can also be used to treat or inhibitcancer. PD-L1 can be overexpressed in tumor cells. Tumor-derived PD-L1can bind to PD-1 on immune cells thereby limiting antitumor T-cellimmunity. Results with small molecule inhibitors, or monoclonalantibodies targeting PD-L1 in murine tumor models, indicate thattargeted PD-L1 therapy is an important alternative and realisticapproach to effective control of tumor growth. As demonstrated in theexperimental examples, the anti-PD-L1 antibodies activated the adaptiveimmune response machinery, which can lead to improved survival in cancerpatients.

Accordingly, in some embodiments, provided are methods for treating acancer in a patient in need thereof. The method, in one embodiment,entails administering to the patient an effective amount of an antibodyof the present disclosure. In some embodiments, at least one of thecancer cells (e.g., stromal cells) in the patient expresses,over-express, or is induced to express PD-L1. Induction of PD-L1expression, for instance, can be done by administration of a tumorvaccine or radiotherapy.

Tumors that express the PD-L1 protein include those of bladder cancer,non-small cell lung cancer, renal cancer, breast cancer, urethralcancer, colorectal cancer, head and neck cancer, squamous cell cancer,Merkel cell carcinoma, gastrointestinal cancer, stomach cancer,oesophageal cancer, ovarian cancer, renal cancer, and small cell lungcancer. Accordingly, the presently disclosed antibodies can be used fortreating any one or more such cancers.

Cellular therapies, such as chimeric antigen receptor (CAR) T-celltherapies, are also provided in the present disclosure. A suitable cellcan be used, that is put in contact with an anti-PD-L1 antibody of thepresent disclosure (or alternatively engineered to express an anti-PD-L1antibody of the present disclosure). Upon such contact or engineering,the cell can then be introduced to a cancer patient in need of atreatment. The cancer patient may have a cancer of any of the types asdisclosed herein. The cell (e.g., T cell) can be, for instance, atumor-infiltrating T lymphocyte, a CD4+ T cell, a CD8+ T cell, or thecombination thereof, without limitation.

In some embodiments, the cell was isolated from the cancer patient him-or her-self. In some embodiments, the cell was provided by a donor orfrom a cell bank. When the cell is isolated from the cancer patient,undesired immune reactions can be minimized.

Additional diseases or conditions associated with increased cellsurvival, that may be treated, prevented, diagnosed and/or prognosedwith the antibodies or variants, or derivatives thereof of thedisclosure include, but are not limited to, progression, and/ormetastases of malignancies and related disorders such as leukemia(including acute leukemias (e.g., acute lymphocytic leukemia, acutemyelocytic leukemia (including myeloblastic, promyelocytic,myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias(e.g., chronic myelocytic (granulocytic) leukemia and chroniclymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin'sdisease and non-Hodgkin's disease), multiple myeloma, Waldenstrom'smacroglobulinemia, heavy chain disease, and solid tumors including, butnot limited to, sarcomas and carcinomas such as fibrosarcoma,myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma,angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyo sarcoma, colon carcinoma, pancreatic cancer,breast cancer, thyroid cancer, endometrial cancer, melanoma, prostatecancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basalcell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma andretinoblastoma.

Combination Therapies

In a further embodiment, the compositions of the disclosure areadministered in combination with an antineoplastic agent, an antiviralagent, antibacterial or antibiotic agent or antifungal agents. Any ofthese agents known in the art may be administered in the compositions ofthe current disclosure.

In another embodiment, compositions of the disclosure are administeredin combination with a chemotherapeutic agent. Chemotherapeutic agentsthat may be administered with the compositions of the disclosureinclude, but are not limited to, antibiotic derivatives (e.g.,doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens(e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU,methotrexate, floxuridine, interferon alpha-2b, glutamic acid,plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g.,carmustine, BCNU, lomustine, CCNU, cytosine arabinoside,cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin,busulfan, cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In an additional embodiment, the compositions of the disclosure areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the disclosure include, but arenot limited to, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13,IL-15, anti-CD40, CD40L, and TNF-α.

In additional embodiments, the compositions of the disclosure areadministered in combination with other therapeutic or prophylacticregimens, such as, for example, radiation therapy.

Combination therapies are also provided, which includes the use of oneor more of the anti-PD-L1 antibody of the present disclosure along witha second anticancer (chemotherapeutic) agent. Chemotherapeutic agentsmay be categorized by their mechanism of action into, for example, thefollowing groups:

anti-metabolites/anti-cancer agents such as pyrimidine analogsfloxuridine, capecitabine, and cytarabine;

purine analogs, folate antagonists, and related inhibitors;

antiproliferative/antimitotic agents including natural products such asvinca alkaloid (vinblastine, vincristine) and microtubule such as taxane(paclitaxel, docetaxel), vinblastin, nocodazole, epothilones,vinorelbine (NAVELBINE®), and epipodophyllotoxins (etoposide,teniposide);

DNA damaging agents such as actinomycin, amsacrine, busulfan,carboplatin, chlorambucil, cisplatin, cyclophosphamide (CYTOXAN®),dactinomycin, daunorubicin, doxorubicin, epirubicin, iphosphamide,melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea,procarbazine, taxol, taxotere, teniposide, etoposide, andtriethylenethiophosphoramide;

antibiotics such as dactinomycin, daunorubicin, doxorubicin, idarubicin,anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), andmitomycin;

enzymes such as L-asparaginase which systemically metabolizesL-asparagine and deprives cells which do not have the capacity tosynthesize their own asparagine;

antiplatelet agents;

antiproliferative/antimitotic alkylating agents such as nitrogenmustards cyclophosphamide and analogs (melphalan, chlorambucil,hexamethylmelamine, and thiotepa), alkyl nitrosoureas (carmustine) andanalogs, streptozocin, and triazenes (dacarbazine);

antiproliferative/antimitotic antimetabolites such as folic acid analogs(methotrexate);

platinum coordination complexes (cisplatin, oxiloplatinim, andcarboplatin), procarbazine, hydroxyurea, mitotane, andaminoglutethimide;

hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide,and nilutamide), and aromatase inhibitors (letrozole and anastrozole);

anticoagulants such as heparin, synthetic heparin salts, and otherinhibitors of thrombin;

fibrinolytic agents such as tissue plasminogen activator, streptokinase,urokinase, aspirin, dipyridamole, ticlopidine, and clopidogrel;

antimigratory agents;

antisecretory agents (breveldin);

immunosuppressives tacrolimus, sirolimus, azathioprine, andmycophenolate;

compounds (TNP-470, genistein) and growth factor inhibitors (vascularendothelial growth factor inhibitors and fibroblast growth factorinhibitors);

angiotensin receptor blockers, nitric oxide donors;

anti-sense oligonucleotides;

antibodies such as trastuzumab and rituximab;

cell cycle inhibitors and differentiation inducers such as tretinoin;

inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin,dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan,mitoxantrone, topotecan, and irinotecan), and corticosteroids(cortisone, dexamethasone, hydrocortisone, methylprednisolone,prednisone, and prednisolone);

growth factor signal transduction kinase inhibitors;

dysfunction inducers;

toxins such as Cholera toxin, ricin, Pseudomonas exotoxin, Bordetellapertussis adenylate cyclase toxin, diphtheria toxin, and caspaseactivators;

and chromatin.

Further examples of chemotherapeutic agents include:

alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®);

alkyl sulfonates such as busulfan, improsulfan, and piposulfan;

aziridines such as benzodopa, carboquone, meturedopa, and uredopa;

emylerumines and memylamelamines including alfretamine,triemylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide, and trimemylolomelamine;

acetogenins, especially bullatacin and bullatacinone;

a camptothecin, including synthetic analog topotecan;

bryostatin;

callystatin;

CC-1065, including its adozelesin, carzelesin, and bizelesin syntheticanalogs;

cryptophycins, particularly cryptophycin 1 and cryptophycin 8;

dolastatin;

duocarmycin, including the synthetic analogs KW-2189 and CBI-TMI;

eleutherobin;

pancratistatin;

a sarcodictyin;

spongistatin;

nitrogen mustards such as chlorambucil, chlornaphazine,cyclophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, and uracil mustard;

nitrosoureas such as carmustine, chlorozotocin, foremustine, lomustine,nimustine, and ranimustine;

antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gammaII and calicheamicin phiI1), dynemicinincluding dynemicin A, bisphosphonates such as clodronate, anesperamicin, neocarzinostatin chromophore and related chromoproteinenediyne antibiotic chromomophores, aclacinomycins, actinomycin,authramycin, azaserine, bleomycins, cactinomycin, carabicin,carrninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin,detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (includingmorpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, and zorubicin;

anti-metabolites such as methotrexate and 5-fluorouracil (5-FU);

folic acid analogs such as demopterin, methotrexate, pteropterin, andtrimetrexate;

purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, andthioguanine;

pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, andfloxuridine;

androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, and testolactone;

anti-adrenals such as aminoglutethimide, mitotane, and trilostane;

folic acid replinishers such as frolinic acid;

trichothecenes, especially T-2 toxin, verracurin A, roridin A, andanguidine;

taxoids such as paclitaxel (TAXOL®) and docetaxel (TAXOTERE®);

platinum analogs such as cisplatin and carboplatin;

aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; hestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformthine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; leucovorin; lonidamine;maytansinoids such as maytansine and ansamitocins; mitoguazone;mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin;losoxantrone; fluoropyrimidine; folinic acid; podophyllinic acid;2-ethylhydrazide; procarbazine; polysaccharide-K (PSK); razoxane;rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-tricUorotriemylamine; urethane; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiopeta; chlorambucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;vinblastine; platinum; etoposide (VP-16); ifosfamide; mitroxantrone;vancristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; xeoloda; ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylomithine (DFMO);retinoids such as retinoic acid; capecitabine; FOLFIRI (fluorouracil,leucovorin, and irinotecan);

and pharmaceutically acceptable salts, acids, or derivatives of any ofthe above.

Also included in the definition of “chemotherapeutic agent” areanti-hormonal agents such as anti-estrogens and selective estrogenreceptor modulators (SERMs), inhibitors of the enzyme aromatase,anti-androgens, and pharmaceutically acceptable salts, acids orderivatives of any of the above that act to regulate or inhibit hormoneaction on tumors.

Examples of anti-estrogens and SERMs include, for example, tamoxifen(including NOLVADEX™), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and toremifene(FARESTON®).

Inhibitors of the enzyme aromatase regulate estrogen production in theadrenal glands. Examples include 4(5)-imidazoles, aminoglutethimide,megestrol acetate (MEGACE®), exemestane, formestane, fadrozole, vorozole(RIVISOR®), letrozole (FEMARA®), and anastrozole (ARIMIDEX®).

Examples of anti-androgens include flutamide, nilutamide, bicalutamide,leuprohde, and goserelin.

Examples of chemotherapeutic agents also include anti-angiogenic agentsincluding, but are not limited to, retinoid acid and derivativesthereof, 2-methoxyestradiol, ANGIOSTATIN®, ENDOSTATIN®, suramin,squalamine, tissue inhibitor of metalloproteinase-1, tissue inhibitor ofmetalloproteinase-2, plasminogen activator inhibitor-1, plasminogenactivator inhibitor-2, cartilage-derived inhibitor, paclitaxel(nab-paclitaxel), platelet factor 4, protamine sulphate (clupeine),sulphated chitin derivatives (prepared from queen crab shells),sulphated polysaccharide peptidoglycan complex (sp-pg), staurosporine,modulators of matrix metabolism including proline analogs((1-azetidine-2-carboxylic acid (LACA)), cishydroxyproline,d,I-3,4-dehydroproline, thiaproline, α,α′-dipyridyl,beta-aminopropionitrile fumarate,4-propyl-5-(4-pyridinyl)-2(3h)-oxazolone, methotrexate, mitoxantrone,heparin, interferons, 2 macroglobulin-serum, chicken inhibitor ofmetalloproteinase-3 (ChIMP-3), chymostatin, beta-cyclodextrintetradecasulfate, eponemycin, fumagillin, gold sodium thiomalate,d-penicillamine, beta-1-anticollagenase-serum, alpha-2-antiplasmin,bisantrene, lobenzarit disodium, n-2-carboxyphenyl-4-chloroanthronilicacid disodium or “CCA”, thalidomide, angiostatic steroid, carboxyaminoimidazole, and metalloproteinase inhibitors such as BB-94. Otheranti-angiogenesis agents include antibodies, preferably monoclonalantibodies against these angiogenic growth factors: beta-FGF, alpha-FGF,FGF-5, VEGF isoforms, VEGF-C, HGF/SF, and Ang-1/Ang-2.

Examples of chemotherapeutic agents also include anti-fibrotic agentsincluding, but are not limited to, the compounds such asbeta-aminoproprionitrile (BAPN), as well as the compounds disclosed inU.S. Pat. No. 4,965,288 (Palfreyman, et al.) relating to inhibitors oflysyl oxidase and their use in the treatment of diseases and conditionsassociated with the abnormal deposition of collagen and U.S. Pat. No.4,997,854 (Kagan et al.) relating to compounds which inhibit LOX for thetreatment of various pathological fibrotic states, which are hereinincorporated by reference. Further exemplary inhibitors are described inU.S. Pat. No. 4,943,593 (Palfreyman et al.) relating to compounds suchas 2-isobutyl-3-fluoro-, chloro-, or bromo-allylamine, U.S. Pat. No.5,021,456 (Palfreyman et al.), U.S. Pat. No. 5,059,714 (Palfreyman etal.), U.S. Pat. No. 5,120,764 (Mccarthy et al.), U.S. Pat. No. 5,182,297(Palfreyman et al.), U.S. Pat. No. 5,252,608 (Palfreyman et al.)relating to 2-(1-naphthyloxymemyl)-3-fluoroallylamine, and U.S. Pub.No.: 2004/0248871 (Farjanel et al.), which are herein incorporated byreference.

Exemplary anti-fibrotic agents also include the primary amines reactingwith the carbonyl group of the active site of the lysyl oxidases, andmore particularly those which produce, after binding with the carbonyl,a product stabilized by resonance, such as the following primary amines:emylenemamine, hydrazine, phenylhydrazine, and their derivatives;semicarbazide and urea derivatives; aminonitriles such as BAPN or2-nitroethylamine; unsaturated or saturated haloamines such as2-bromo-ethylamine, 2-chloroethylamine, 2-trifluoroethylamine,3-bromopropylamine, and p-halobenzylamines; and selenohomocysteinelactone.

Other anti-fibrotic agents are copper chelating agents penetrating ornot penetrating the cells. Exemplary compounds include indirectinhibitors which block the aldehyde derivatives originating from theoxidative deamination of the lysyl and hydroxylysyl residues by thelysyl oxidases. Examples include the thiolamines, particularlyD-penicillamine, and its analogs such as2-amino-5-mercapto-5-methylhexanoic acid,D-2-amino-3-methyl-3-((2-acetamidoethyl)dithio)butanoic acid,p-2-amino-3-methyl-3-((2-aminoethyl)dithio)butanoic acid,sodium-4-((p-1-dimethyl-2-amino-2-carboxyethyl)dithio)butane sulphurate,2-acetamidoethyl-2-acetamidoethanethiol sulphanate, andsodium-4-mercaptobutanesulphinate trihydrate.

Examples of chemotherapeutic agents also include immunotherapeuticagents including and are not limited to therapeutic antibodies suitablefor treating patients. Some examples of therapeutic antibodies includesimtuzumab, abagovomab, adecatumumab, afutuzumab, alemtuzumab,altumomab, amatuximab, anatumomab, arcitumomab, bavituximab, bectumomab,bevacizumab, bivatuzumab, blinatumomab, brentuximab, cantuzumab,catumaxomab, cetuximab, citatuzumab, cixutumumab, clivatuzumab,conatumumab, daratumumab, drozitumab, duligotumab, dusigitumab,detumomab, dacetuzumab, dalotuzumab, ecromeximab, elotuzumab,ensituximab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab,figitumumab, flanvotumab, futuximab, ganitumab, gemtuzumab,girentuximab, glembatumumab, ibritumomab, igovomab, imgatuzumab,indatuximab, inotuzumab, intetumumab, ipilimumab, iratumumab,labetuzumab, lexatumumab, lintuzumab, lorvotuzumab, lucatumumab,mapatumumab, matuzumab, milatuzumab, minretumomab, mitumomab,moxetumomab, narnatumab, naptumomab, necitumumab, nimotuzumab,nofetumomab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab,oportuzumab, oregovomab, panitumumab, parsatuzumab, patritumab,pemtumomab, pertuzumab, pintumomab, pritumumab, racotumomab, radretumab,rilotumumab, rituximab, robatumumab, satumomab, sibrotuzumab,siltuximab, solitomab, tacatuzumab, taplitumomab, tenatumomab,teprotumumab, tigatuzumab, tositumomab, trastuzumab, tucotuzumab,ublituximab, veltuzumab, vorsetuzumab, votumumab, zalutumumab, CC49, and3F8. Rituximab can be used for treating indolent B-cell cancers,including marginal-zone lymphoma, WM, CLL and small lymphocyticlymphoma. A combination of Rituximab and chemotherapy agents isespecially effective.

The exemplified therapeutic antibodies may be further labeled orcombined with a radioisotope particle such as indium-111, yttrium-90, oriodine-131.

In a one embodiment, the additional therapeutic agent is a nitrogenmustard alkylating agent. Nonlimiting examples of nitrogen mustardalkylating agents include chlorambucil.

In one embodiment, the compounds and compositions described herein maybe used or combined with one or more additional therapeutic agents. Theone or more therapeutic agents include, but are not limited to, aninhibitor of Abl, activated CDC kinase (ACK), adenosine A2B receptor(A2B), apoptosis signal-regulating kinase (ASK), Auroa kinase, Bruton'styrosine kinase (BTK), BET-bromodomain (BRD) such as BRD4, c-Kit, c-Met,CDK-activating kinase (CAK), calmodulin-dependent protein kinase (CaMK),cyclin-dependent kinase (CDK), casein kinase (CK), discoidin domainreceptor (DDR), epidermal growth factor receptors (EGFR), focal adhesionkinase (FAK), Flt-3, FYN, glycogen synthase kinase (GSK), HCK, histonedeacetylase (HDAC), IKK such as IKKIβε, isocitrate dehydrogenase (IDH)such as IDH1, Janus kinase (JAK), KDR, lymphocyte-specific proteintyrosine kinase (LCK), lysyl oxidase protein, lysyl oxidase-like protein(LOXL), LYN, matrix metalloprotease (MMP), MEK, mitogen-activatedprotein kinase (MAPK), NEK9, NPM-ALK, p38 kinase, platelet-derivedgrowth factor (PDGF), phosphorylase kinase (PK), polo-like kinase (PLK),phosphatidylinositol 3-kinase (PI3K), protein kinase (PK) such asprotein kinase A, B, and/or C, PYK, spleen tyrosine kinase (SYK),serine/threonine kinase TPL2, serine/threonine kinase STK, signaltransduction and transcription (STAT), SRC, serine/threonine-proteinkinase (TBK) such as TBK1, TIE, tyrosine kinase (TK), vascularendothelial growth factor receptor (VEGFR), YES, or any combinationthereof.

ASK inhibitors include ASK1 inhibitors. Examples of ASK1 inhibitorsinclude, but are not limited to, those described in WO 2011/008709(Gilead Sciences) and WO 2013/112741 (Gilead Sciences).

Examples of BTK inhibitors include, but are not limited to, ibrutinib,HM71224, ONO-4059, and CC-292.

DDR inhibitors include inhibitors of DDR1 and/or DDR2. Examples of DDRinhibitors include, but are not limited to, those disclosed in WO2014/047624 (Gilead Sciences), US 2009/0142345 (Takeda Pharmaceutical),US 2011/0287011 (Oncomed Pharmaceuticals), WO 2013/027802 (ChugaiPharmaceutical), and WO 2013/034933 (Imperial Innovations).

Examples of HDAC inhibitors include, but are not limited to, pracinostatand panobinostat.

JAK inhibitors inhibit JAK1, JAK2, and/or JAK3. Examples of JAKinhibitors include, but are not limited to, filgotinib, ruxolitinib,fedratinib, tofacitinib, baricitinib, lestaurtinib, pacritinib, XL019,AZD1480, INCB039110, LY2784544, BMS911543, and NS018.

LOXL inhibitors include inhibitors of LOXL1, LOXL2, LOXL3, LOXL4, and/orLOXL5. Examples of LOXL inhibitors include, but are not limited to, theantibodies described in WO 2009/017833 (Arresto Biosciences).

Examples of LOXL2 inhibitors include, but are not limited to, theantibodies described in WO 2009/017833 (Arresto Biosciences), WO2009/035791 (Arresto Biosciences), and WO 2011/097513 (GileadBiologics).

MMP inhibitors include inhibitors of MMP1 through 10. Examples of MMP9inhibitors include, but are not limited to, marimastat (BB-2516),cipemastat (Ro 32-3555), and those described in WO 2012/027721 (GileadBiologics).

PI3K inhibitors include inhibitors of PI3Kγ, PI3Kδ, PI3Kβ, PI3Kα, and/orpan-PI3K. Examples of PI3K inhibitors include, but are not limited to,wortmannin, BKM120, CH5132799, XL756, and GDC-0980.

Examples of PI3Kγ inhibitors include, but are not limited to, ZSTK474,AS252424, LY294002, and TG100115.

Examples of PI3Kδ inhibitors include, but are not limited to, PI3K II,TGR-1202, AMG-319, GSK2269557, X-339, X-414, RP5090, KAR4141, XL499,OXY111A, IPI-145, IPI-443, and the compounds described in WO 2005/113556(ICOS), WO 2013/052699 (Gilead Calistoga), WO 2013/116562 (GileadCalistoga), WO 2014/100765 (Gilead Calistoga), WO 2014/100767 (GileadCalistoga), and WO 2014/201409 (Gilead Sciences).

Examples of PI3Kβ inhibitors include, but are not limited to,GSK2636771, BAY 10824391, and TGX221.

Examples of PI3Kα inhibitors include, but are not limited to,buparlisib, BAY 80-6946, BYL719, PX-866, RG7604, MLN1117, WX-037,AEZA-129, and PA799.

Examples of pan-PI3K inhibitors include, but are not limited to,LY294002, BEZ235, XL147 (SAR245408), and GDC-0941.

Examples of SYK inhibitors include, but are not limited to, tamatinib(R406), fostamatinib (R788), PRT062607, BAY-61-3606, NVP-QAB 205 AA,R112, R343, and those described in U.S. Pat. No. 8,450,321 (GileadConnecticut).

TKIs may target epidermal growth factor receptors (EGFRs) and receptorsfor fibroblast growth factor (FGF), platelet-derived growth factor(PDGF), and vascular endothelial growth factor (VEGF). Examples of TKIsthat target EGFR include, but are not limited to, gefitinib anderlotinib. Sunitinib is a non-limiting example of a TKI that targetsreceptors for FGF, PDGF, and VEGF.

The anti-PD-L1 antibodies of the present disclosure can be used, in someembodiments, together with an immune checkpoint inhibitor. Immunecheckpoints are molecules in the immune system that either turn up asignal (co-stimulatory molecules) or turn down a signal (co-inhibitorymolecules). Many cancers protect themselves from the immune system byinhibiting the T cell signal through agonist for co-inhibitory moleculesor antagonist for co-stimulatory molecules. An immune checkpoint agonistor antagonist can help stop such a protective mechanism by the cellcells. An immune checkpoint agonist or antagonistmay target any one ormore of the following checkpoint molecules, PD-1, CTLA-4, LAG-3 (alsoknown as CD223), CD28, CD122, 4-1BB (also known as CD137), TIM3,OX-40/OX40L, CD40/CD40L, LIGHT, ICOS/ICOSL, GITR/GITRL, TIGIT, CD27,VISTA, B7H3, B7H4, HEVM or BTLA (also known as CD272).

Programmed T cell death 1 (PD-1) is a trans-membrane protein found onthe surface of T cells, which, when bound to programmed T cell deathligand 1 (PD-L1) on tumor cells, results in suppression of T cellactivity and reduction of T cell-mediated cytotoxicity. Thus, PD-1 andPD-L1 are immune down-regulators or immune checkpoint “off switches”.Example PD-1 inhibitor include, without limitation, nivolumab, (Opdivo)(BMS-936558), pembrolizumab (Keytruda), pidilizumab, AMP-224, MEDI0680(AMP-514), PDR001, MPDL3280A, MEDI4736, BMS-936559 and MSB0010718C.

CTLA-4 is a protein receptor that downregulates the immune system.Non-limiting examples of CTLA-4 inhibitors include ipilimumab (Yervoy)(also known as BMS-734016, MDX-010, MDX-101) and tremelimumab (formerlyticilimumab, CP-675,206).

Lymphocyte-activation gene 3 (LAG-3) is an immune checkpoint receptor onthe cell surface works to suppress an immune response by action to Tregsas well as direct effects on CD8+ T cells. LAG-3 inhibitors include,without limitation, LAG525 and BMS-986016.

CD28 is constitutively expressed on almost all human CD4+ T cells and onaround half of all CD8 T cells prompts T cell expansion. Non-limitingexamples of CD28 inhibitors include TGN1412.

CD122 increases the proliferation of CD8+ effector T cells. Non-limitingexamples include NKTR-214.

4-1BB (also known as CD137) is involved in T-cell proliferation.CD137-mediated signaling is also known to protect T cells, and inparticular, CD8+ T cells from activation-induced cell death.PF-05082566, Urelumab (BMS-663513) and lipocalin are example CD137inhibitors.

For any of the above combination treatments, the anti-PD-L1 antibody canbe administered concurrently or separately from the other anticanceragent. When administered separately, the anti-PD-L1 antibody can beadministered before or after the other anticancer agent.

Treatment of Infections

As demonstrated in the experimental examples, the antibodies of thepresent disclosure can activate immune response which can then be usefulfor treating infections.

Infection is the invasion of an organism's body tissues bydisease-causing agents, their multiplication, and the reaction of hosttissues to these organisms and the toxins they produce. An infection canbe caused by infectious agents such as viruses, viroids, prions,bacteria, nematodes such as parasitic roundworms and pinworms,arthropods such as ticks, mites, fleas, and lice, fungi such asringworm, and other macroparasites such as tapeworms and otherhelminths. In one aspect, the infectious agent is a bacterium, such asGram negative bacterium. In one aspect, the infectious agent is virus,such as DNA viruses, RNA viruses, and reverse transcribing viruses.Non-limiting examples of viruses include Adenovirus, Coxsackievirus,Epstein-Barr virus, Hepatitis A virus, Hepatitis B virus, Hepatitis Cvirus, Herpes simplex virus, type 1, Herpes simplex virus, type 2,Cytomegalovirus, Human herpesvirus, type 8, HIV, Influenza virus,Measles virus, Mumps virus, Human papillomavirus, Parainfluenza virus,Poliovirus, Rabies virus, Respiratory syncytial virus, Rubella virus,Varicella-zoster virus.

The antibodies of the present disclosure can also be used to treat aninfectious disease caused by a microorganism, or kill a microorganism,by targeting the microorganism and an immune cell to effect eliminationof the microorganism. In one aspect, the microorganism is a virusincluding RNA and DNA viruses, a Gram positive bacterium, a Gramnegative bacterium, a protozoa or a fungus. Non-limiting examples ofinfectious diseases and related microorganisms are provided in Table 4below.

TABLE 4 Infectious diseases and related microorganism sources.Infectious Disease Microorganism Source Acinetobacter infectionsAcinetobacter baumannii Actinomycosis Actinomyces israelii, Actinomycesgerencseriae and Propionibacterium propionicus African sleeping sickness(African Trypanosoma brucei trypanosomiasis) AIDS (Acquiredimmunodeficiency HIV (Human immunodeficiency virus) syndrome) AmebiasisEntamoeba histolytica Anaplasmosis Anaplasma genus Anthrax Bacillusanthracis Arcanobacterium haemolyticum Arcanobacterium haemolyticuminfection Argentine hemorrhagic fever Junin virus Ascariasis Ascarislumbricoides Aspergillosis Aspergillus genus Astrovirus infectionAstroviridae family Babesiosis Babesia genus Bacillus cereus infectionBacillus cereus Bacterial pneumonia multiple bacteria Bacterialvaginosis (BV) multiple bacteria Bacteroides infection Bacteroides genusBalantidiasis Balantidium coli Baylisascaris infection Baylisascarisgenus BK virus infection BK virus Black piedra Piedraia hortaeBlastocystis hominis infection Blastocystis hominis BlastomycosisBlastomyces dermatitidis Bolivian hemorrhagic fever Machupo virusBorrelia infection Borrelia genus Botulism (and Infant botulism)Clostridium botulinum Brazilian hemorrhagic fever Sabia BrucellosisBrucella genus Burkholderia infection usually Burkholderia cepacia andother Burkholderia species Buruli ulcer Mycobacterium ulceransCalicivirus infection (Norovirus and Caliciviridae family Sapovirus)Campylobacteriosis Campylobacter genus Candidiasis (Moniliasis; Thrush)usually Candida albicans and other Candida species Cat-scratch diseaseBartonella henselae Cellulitis usually Group A Streptococcus andStaphylococcus Chagas Disease (American Trypanosoma cruzitrypanosomiasis) Chancroid Haemophilus ducreyi Chickenpox Varicellazoster virus (VZV) Chlamydia Chlamydia trachomatis Chlamydophilapneumoniae infection Chlamydophila pneumoniae Cholera Vibrio choleraeChromoblastomycosis usually Fonsecaea pedrosoi Clonorchiasis Clonorchissinensis Clostridium difficile infection Clostridium difficileCoccidioidomycosis Coccidioides immitis and Coccidioides posadasiiColorado tick fever (CTF) Colorado tick fever virus (CTFV) Common cold(Acute viral usually rhinoviruses and coronaviruses. rhinopharyngitis;Acute coryza) Creutzfeldt-Jakob disease (CJD) CJD prion Crimean-Congohemorrhagic fever Crimean-Congo hemorrhagic fever virus (CCHF)Cryptococcosis Cryptococcus neoformans Cryptosporidiosis Cryptosporidiumgenus Cutaneous larva migrans (CLM) usually Ancylostoma braziliense;multiple other parasites Cyclosporiasis Cyclospora cayetanensisCysticercosis Taenia solium Cytomegalovirus infection CytomegalovirusDengue fever Dengue viruses (DEN-1, DEN-2, DEN-3 and DEN-4) -Flaviviruses Dientamoebiasis Dientamoeba fragilis DiphtheriaCorynebacterium diphtheriae Diphyllobothriasis DiphyllobothriumDracunculiasis Dracunculus medinensis Ebola hemorrhagic fever Ebolavirus(EBOV) Echinococcosis Echinococcus genus Ehrlichiosis Ehrlichia genusEnterobiasis (Pinworm infection) Enterobius vermicularis Enterococcusinfection Enterococcus genus Enterovirus infection Enterovirus genusEpidemic typhus Rickettsia prowazekii Erythema infectiosum (Fifthdisease) Parvovirus B19 Exanthem subitum (Sixth disease) Humanherpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV-7) FasciolopsiasisFasciolopsis buski Fasciolosis Fasciola hepatica and Fasciola giganticaFatal familial insomnia (FFI) FFI prion Filariasis Filarioideasuperfamily Food poisoning by Clostridium Clostridium perfringensperfringens Free-living amebic infection multiple Fusobacteriuminfection Fusobacterium genus Gas gangrene (Clostridial usuallyClostridium perfringens; other Clostridium myonecrosis) speciesGeotrichosis Geotrichum candidum Gerstmann-Sträussler-Scheinker GSSprion syndrome (GSS) Giardiasis Giardia intestinalis GlandersBurkholderia mallei Gnathostomiasis Gnathostoma spinigerum andGnathostoma hispidum Gonorrhea Neisseria gonorrhoeae Granuloma inguinale(Donovanosis) Klebsiella granulomatis Group A streptococcal infectionStreptococcus pyogenes Group B streptococcal infection Streptococcusagalactiae Haemophilus influenzae infection Haemophilus influenzae Hand,foot and mouth disease Enteroviruses, mainly Coxsackie A virus andEnterovirus (HFMD) 71 (EV71) Hantavirus Pulmonary Syndrome Sin Nombrevirus (HPS) Helicobacter pylori infection Helicobacter pyloriHemolytic-uremic syndrome (HUS) Escherichia coli O157:H7, O111 andO104:H4 Hemorrhagic fever with renal Bunyaviridae family syndrome (HFRS)Hepatitis A Hepatitis A Virus Hepatitis B Hepatitis B Virus Hepatitis CHepatitis C Virus Hepatitis D Hepatitis D Virus Hepatitis E Hepatitis EVirus Herpes simplex Herpes simplex virus 1 and 2 (HSV-1 and HSV-2)Histoplasmosis Histoplasma capsulatum Hookworm infection Ancylostomaduodenale and Necator americanus Human bocavirus infection Humanbocavirus (HBoV) Human ewingii ehrlichiosis Ehrlichia ewingii Humangranulocytic anaplasmosis Anaplasma phagocytophilum (HGA) Humanmetapneumovirus infection Human metapneumovirus (hMPV) Human monocyticehrlichiosis Ehrlichia chaffeensis Human papillomavirus (HPV) Humanpapillomavirus (HPV) infection Human parainfluenza virus infection Humanparainfluenza viruses (HPIV) Hymenolepiasis Hymenolepis nana andHymenolepis diminuta Epstein-Barr Virus Infectious Epstein-Barr Virus(EBV) Mononucleosis (Mono) Influenza (flu) Orthomyxoviridae familyIsosporiasis Isospora belli Kawasaki disease unknown; evidence supportsthat it is infectious Keratitis multiple Kingella kingae infectionKingella kingae Kuru Kuru prion Lassa fever Lassa virus Legionellosis(Legionnaires' disease) Legionella pneumophila Legionellosis (Pontiacfever) Legionella pneumophila Leishmaniasis Leishmania genus LeprosyMycobacterium leprae and Mycobacterium lepromatosis LeptospirosisLeptospira genus Listeriosis Listeria monocytogenes Lyme disease (Lymeborreliosis) usually Borrelia burgdorferi and other Borrelia speciesLymphatic filariasis (Elephantiasis) Wuchereria bancrofti and Brugiamalayi Lymphocytic choriomeningitis Lymphocytic choriomeningitis virus(LCMV) Malaria Plasmodium genus Marburg hemorrhagic fever (MHF) Marburgvirus Measles Measles virus Melioidosis (Whitmore's disease)Burkholderia pseudomallei Meningitis multiple Meningococcal diseaseNeisseria meningitidis Metagonimiasis usually Metagonimus yokagawaiMicrosporidiosis Microsporidia phylum Molluscum contagiosum (MC)Molluscum contagiosum virus (MCV) Mumps Mumps virus Murine typhus(Endemic typhus) Rickettsia typhi Mycoplasma pneumonia Mycoplasmapneumoniae Mycetoma numerous species of bacteria (Actinomycetoma) andfungi (Eumycetoma) Myiasis parasitic dipterous fly larvae Neonatalconjunctivitis (Ophthalmia most commonly Chlamydia trachomatis andNeisseria neonatorum) gonorrhoeae (New) Variant Creutzfeldt-Jakob vCJDprion disease (vCJD, nvCJD) Nocardiosis usually Nocardia asteroides andother Nocardia species Onchocerciasis (River blindness) Onchocercavolvulus Paracoccidioidomycosis (South Paracoccidioides brasiliensisAmerican blastomycosis) Paragonimiasis usually Paragonimus westermaniand other Paragonimus species Pasteurellosis Pasteurella genusPediculosis capitis (Head lice) Pediculus humanus capitis Pediculosiscorporis (Body lice) Pediculus humanus corporis Pediculosis pubis (Pubiclice, Crab Phthirus pubis lice) Pelvic inflammatory disease (PID)multiple Pertussis (Whooping cough) Bordetella pertussis Plague Yersiniapestis Pneumococcal infection Streptococcus pneumoniae Pneumocystispneumonia (PCP) Pneumocystis jirovecii Pneumonia multiple PoliomyelitisPoliovirus Prevotella infection Prevotella genus Primary amoebic usuallyNaegleria fowleri meningoencephalitis (PAM) Progressive multifocal JCvirus leukoencephalopathy Psittacosis Chlamydophila psittaci Q feverCoxiella burnetii Rabies Rabies virus Rat-bite fever Streptobacillusmoniliformis and Spirillum minus Respiratory syncytial virus infectionRespiratory syncytial virus (RSV) Rhinosporidiosis Rhinosporidiumseeberi Rhinovirus infection Rhinovirus Rickettsial infection Rickettsiagenus Rickettsialpox Rickettsia akari Rift Valley fever (RVF) RiftValley fever virus Rocky mountain spotted fever Rickettsia rickettsii(RMSF) Rotavirus infection Rotavirus Rubella Rubella virus SalmonellosisSalmonella genus SARS (Severe Acute Respiratory SARS coronavirusSyndrome) Scabies Sarcoptes scabiei Schistosomiasis Schistosoma genusSepsis multiple Shigellosis (Bacillary dysentery) Shigella genusShingles (Herpes zoster) Varicella zoster virus (VZV) Smallpox (Variola)Variola major or Variola minor Sporotrichosis Sporothrix schenckiiStaphylococcal food poisoning Staphylococcus genus Staphylococcalinfection Staphylococcus genus Strongyloidiasis Strongyloidesstercoralis Syphilis Treponema pallidum Taeniasis Taenia genus Tetanus(Lockjaw) Clostridium tetani Tinea barbae (Barber's itch) usuallyTrichophyton genus Tinea capitis (Ringworm of the Scalp) usuallyTrichophyton tonsurans Tinea corporis (Ringworm of the usuallyTrichophyton genus Body) Tinea cruris (Jock itch) usually Epidermophytonfloccosum, Trichophyton rubrum, and Trichophyton mentagrophytes Tineamanuum (Ringworm of the Trichophyton rubrum Hand) Tinea nigra usuallyHortaea werneckii Tinea pedis (Athlete's foot) usually Trichophytongenus Tinea unguium (Onychomycosis) usually Trichophyton genus Tineaversicolor (Pityriasis Malassezia genus versicolor) Toxocariasis (OcularLarva Migrans Toxocara canis or Toxocara cati (OLM)) Toxocariasis(Visceral Larva Migrans Toxocara canis or Toxocara cati (VLM))Toxoplasmosis Toxoplasma gondii Trichinellosis Trichinella spiralisTrichomoniasis Trichomonas vaginalis Trichuriasis (Whipworm infection)Trichuris trichiura Tuberculosis usually Mycobacterium tuberculosisTularemia Francisella tularensis Ureaplasma urealyticum infectionUreaplasma urealyticum Venezuelan equine encephalitis Venezuelan equineencephalitis virus Venezuelan hemorrhagic fever Guanarito virus Viralpneumonia multiple viruses West Nile Fever West Nile virus White piedra(Tinea blanca) Trichosporon beigelii Yersinia pseudotuberculosisinfection Yersinia pseudotuberculosis Yersiniosis Yersiniaenterocolitica Yellow fever Yellow fever virus Zygomycosis Mucoralesorder (Mucormycosis) and Entomophthorales order (Entomophthoramycosis)

A specific dosage and treatment regimen for any particular patient willdepend upon a variety of factors, including the particular antibodies,variant or derivative thereof used, the patient's age, body weight,general health, sex, and diet, and the time of administration, rate ofexcretion, drug combination, and the severity of the particular diseasebeing treated. Judgment of such factors by medical caregivers is withinthe ordinary skill in the art. The amount will also depend on theindividual patient to be treated, the route of administration, the typeof formulation, the characteristics of the compound used, the severityof the disease, and the desired effect. The amount used can bedetermined by pharmacological and pharmacokinetic principles well knownin the art.

Methods of administration of the antibodies, variants or include but arenot limited to intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, and oral routes. The antigen-bindingpolypeptides or compositions may be administered by any convenientroute, for example by infusion or bolus injection, by absorption throughepithelial or mucocutaneous linings (e.g., oral mucosa, rectal andintestinal mucosa, etc.) and may be administered together with otherbiologically active agents. Thus, pharmaceutical compositions containingthe antigen-binding polypeptides of the disclosure may be administeredorally, rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, drops ortransdermal patch), bucally, or as an oral or nasal spray.

The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intra-articular injection and infusion.

Administration can be systemic or local. In addition, it may bedesirable to introduce the antibodies of the disclosure into the centralnervous system by any suitable route, including intraventricular andintrathecal injection; intraventricular injection may be facilitated byan intraventricular catheter, for example, attached to a reservoir, suchas an Ommaya reservoir. Pulmonary administration can also be employed,e.g., by use of an inhaler or nebulizer, and formulation with anaerosolizing agent.

It may be desirable to administer the antibodies polypeptides orcompositions of the disclosure locally to the area in need of treatment;this may be achieved by, for example, and not by way of limitation,local infusion during surgery, topical application, e.g., inconjunction, with a wound dressing after surgery, by injection, by meansof a catheter, by means of a suppository, or by means of an implant,said implant being of a porous, non-porous, or gelatinous material,including membranes, such as sialastic membranes, or fibers. Preferably,when administering a protein, including an antibody, of the disclosure,care must be taken to use materials to which the protein does notabsorb.

In another embodiment, the antibodies or composition can be delivered ina vesicle, in particular a liposome (see Langer, 1990, Science249:1527-1533; Treat et al., in Liposomes in the Therapy of InfectiousDisease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, N.Y., pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the antigen-binding polypeptide orcomposition can be delivered in a controlled release system. In oneembodiment, a pump may be used (see Sefton, 1987, CRC Crit. Ref. Biomed.Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989,N. Engl. J. Med. 321:574). In another embodiment, polymeric materialscan be used (see Medical Applications of Controlled Release, Langer andWise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol.Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989,J. Neurosurg. 71:105). In yet another embodiment, a controlled releasesystem can be placed in proximity of the therapeutic target, i.e., thebrain, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, vol. 2,pp. 115-138 (1984)). Other controlled release systems are discussed inthe review by Langer (1990, Science 249:1527-1533).

In a specific embodiment where the composition of the disclosurecomprises a nucleic acid or polynucleotide encoding a protein, thenucleic acid can be administered in vivo to promote expression of itsencoded protein, by constructing it as part of an appropriate nucleicacid expression vector and administering it so that it becomesintracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by use of microparticlebombardment (e.g., a gene gun; Biolistic, Dupont), or coating withlipids or cell-surface receptors or transfecting agents, or byadministering it in linkage to a homeobox-like peptide which is known toenter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad.Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The amount of the antibodies of the disclosure which will be effectivein the treatment, inhibition and prevention of an inflammatory, immuneor malignant disease, disorder or condition can be determined bystandard clinical techniques. In addition, in vitro assays mayoptionally be employed to help identify optimal dosage ranges. Theprecise dose to be employed in the formulation will also depend on theroute of administration, and the seriousness of the disease, disorder orcondition, and should be decided according to the judgment of thepractitioner and each patient's circumstances. Effective doses may beextrapolated from dose-response curves derived from in vitro or animalmodel test systems.

As a general proposition, the dosage administered to a patient of theantigen-binding polypeptides of the present disclosure is typically 0.1mg/kg to 100 mg/kg of the patient's body weight, between 0.1 mg/kg and20 mg/kg of the patient's body weight, or 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of thedisclosure may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The methods for treating an infectious or malignant disease, conditionor disorder comprising administration of an antibody, variant, orderivative thereof of the disclosure are typically tested in vitro, andthen in vivo in an acceptable animal model, for the desired therapeuticor prophylactic activity, prior to use in humans. Suitable animalmodels, including transgenic animals, are well known to those ofordinary skill in the art. For example, in vitro assays to demonstratethe therapeutic utility of antigen-binding polypeptide described hereininclude the effect of an antigen-binding polypeptide on a cell line or apatient tissue sample. The effect of the antigen-binding polypeptide onthe cell line and/or tissue sample can be determined utilizingtechniques known to those of skill in the art, such as the assaysdisclosed elsewhere herein. In accordance with the disclosure, in vitroassays which can be used to determine whether administration of aspecific antigen-binding polypeptide is indicated, include in vitro cellculture assays in which a patient tissue sample is grown in culture, andexposed to or otherwise administered a compound, and the effect of suchcompound upon the tissue sample is observed.

Various delivery systems are known and can be used to administer anantibody of the disclosure or a polynucleotide encoding an antibody ofthe disclosure, e.g., encapsulation in liposomes, microparticles,microcapsules, recombinant cells capable of expressing the compound,receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol.Chem. 262:4429-4432), construction of a nucleic acid as part of aretroviral or other vector, etc.

Diagnostic Methods

Over-expression of PD-L1 is observed in certain tumor samples, andpatients having PD-L1-over-expressing cells are likely responsive totreatments with the anti-PD-L1 antibodies of the present disclosure.Accordingly, the antibodies of the present disclosure can also be usedfor diagnostic and prognostic purposes.

A sample that preferably includes a cell can be obtained from a patient,which can be a cancer patient or a patient desiring diagnosis. The cellbe a cell of a tumor tissue or a tumor block, a blood sample, a urinesample or any sample from the patient. Upon optional pre-treatment ofthe sample, the sample can be incubated with an antibody of the presentdisclosure under conditions allowing the antibody to interact with aPD-L1 protein potentially present in the sample. Methods such as ELISAcan be used, taking advantage of the anti-PD-L1 antibody, to detect thepresence of the PD-L1 protein in the sample.

Presence of the PD-L1 protein in the sample (optionally with the amountor concentration) can be used for diagnosis of cancer, as an indicationthat the patient is suitable for a treatment with the antibody, or as anindication that the patient has (or has not) responded to a cancertreatment. For a prognostic method, the detection can be done at once,twice or more, at certain stages, upon initiation of a cancer treatmentto indicate the progress of the treatment.

Compositions

The present disclosure also provides pharmaceutical compositions. Suchcompositions comprise an effective amount of an antibody, and anacceptable carrier. In some embodiments, the composition furtherincludes a second anticancer agent (e.g., an immune checkpointinhibitor).

In a specific embodiment, the term “pharmaceutically acceptable” meansapproved by a regulatory agency of the Federal or a state government orlisted in the U.S. Pharmacopeia or other generally recognizedpharmacopeia for use in animals, and more particularly in humans.Further, a “pharmaceutically acceptable carrier” will generally be anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type.

The term “carrier” refers to a diluent, adjuvant, excipient, or vehiclewith which the therapeutic is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a preferredcarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions can also be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene, glycol, water, ethanol and thelike. The composition, if desired, can also contain minor amounts ofwetting or emulsifying agents, or pH buffering agents such as acetates,citrates or phosphates. Antibacterial agents such as benzyl alcohol ormethyl parabens; antioxidants such as ascorbic acid or sodium bisulfate;chelating agents such as ethylenediaminetetraacetic acid; and agents forthe adjustment of tonicity such as sodium chloride or dextrose are alsoenvisioned. These compositions can take the form of solutions,suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like. The composition can beformulated as a suppository, with traditional binders and carriers suchas triglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences by E. W. Martin, incorporated herein byreference. Such compositions will contain a therapeutically effectiveamount of the antigen-binding polypeptide, preferably in purified form,together with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

In an embodiment, the composition is formulated in accordance withroutine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the disclosure can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

EXAMPLES Example 1: Generation of Human Monoclonal Antibodies AgainstHuman PD-L1

Anti-human-PD-L1 mouse monoclonal antibodies were generated using thehybridoma technology.

Antigen: human PDL1-Fc protein and human PD-L1 highly expressed CHOK1cell line (PDL1-CHOK1 cell line).

Immunization: To generate mouse monoclonal antibodies to human PD-L1,6-8 week female BALB/c mice were firstly immunized with 1.5×10⁷PDL1-CHOK1 cells. Day 14 and 33 post first immunization, the immunizedmice were re-immunized with 1.5×10⁷ PDL1-CHOK1 cells respectively. Toselect mice producing antibodies that bond PD-L1 protein, sera fromimmunized mice were tested by ELISA. Briefly, microtiter plates werecoated with human PD-L1 protein at 1 μg/ml in PBS, 100 μl/well at roomtemperature (RT) overnight, then blocked with 100 μl/well of 5% BSA.Dilutions of plasma from immunized mice were added to each well andincubated for 1-2 hours at RT. The plates were washed with PBS/Tween andthen incubate with anti-mouse IgG antibody conjugated with Horse RadishPeroxidase (HRP) for 1 hour at RT. After washing, the plates weredeveloped with ABTS substrate and analyzed by spectrophotometer at OD405 nm. Mice with sufficient titers of anti-PDL1 IgG were boosted with50 ug human PDL1-Fc protein at Day 54 post-immunization. The resultingmice were used for fusions. The hybridoma supernatants were tested foranti-PD-L1 IgGs by ELISA.

Hybridoma clones HL1210-3, HL1207-3, HL1207-9 and HL1120-3 were selectedfor further analysis. The amino acid and polynucleotide sequences of thevariable regions of HL1210-3 are provided in Table 5 below.

TABLE 5  HL1210-3 variable sequences SEQ Name Sequence ID NO: HL1210-3GAAGTGAAACTGGTGGAGTCTGGGGGAGACTTAGT 112 VHGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAG CCTCTGGATTCACTTTCAGTAGCTATGACATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGTCTGGAGTG GGTCGCAACCATTAGTGATGGTGGTGGTTACATCTACTATTCAGACAGTGTGAAGGGGCGATTTACCATC TCCAGAGACAATGCCAAGAACAACCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCTTGT ATATTTGTGCAAGAGAATTTGGTAAGCGCTATGCTTTGGACTACTGGGGTCAAGGAACCTCAGTCACCGT CTCCTCA HL1210-3EVKLVESGGDLVKPGGSLKLSCAASGFTFSSYDMS 113 VHWVRQTPEKSLEWVATISDGGGYIYYSDSVKGRFTI SRDNAKNNLYLQMSSLRSEDTALYICAREFGKRYALDYWGQGTSVT HL1210-3 GACATTGTGATGACCCAGTCTCACAAATTCATGTC 114 VLCACATCGGTAGGAGACAGGGTCAGCATCTCCTGCA AGGCCAGTCAGGATGTGACTCCTGCTGTCGCCTGGTATCAACAGAAGCCAGGACAATCTCCTAAACTACT GATTTACTCCACATCCTCCCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGAT TTCACTTTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATACTA CTCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA HL1210-3 DIVMTQSHKFMSTSVGDRVSISCKASQDVTPAVAW 115 VLYQQKPGQSPKLLIYSTSSRYTGVPDRFTGSGSGTD FTFTISSVQAEDLAVYYCQQHYTTPLTFGAGTKLELK

Example 2: HL1210-3 Mouse Monoclonal Antibody's Binding Activity forHuman PD-L1

To evaluate the binding activity of hybridoma clone HL1210-3, thepurified mAb from this clone were subjected to ELISA test. Briefly,microtiter plates were coated with human PD-L1-Fc protein at 0.1 μg/mlin PBS, 100 μl/well at 4° C. overnight, then blocked with 100 μl/well of5% BSA. Three-fold dilutions of HL1210-3 antibodies starting from 0.2μg/ml were added to each well and incubated for 1-2 hours at RT. Theplates were washed with PBS/Tween and then incubate with goat-anti-mouseIgG antibody conjugated with Horse Radish Peroxidase (HRP) for 1 hour atRT. After washing, the plates were developed with TMB substrate andanalyzed by spectrophotometer at OD 450-630 nm. As shown in FIG. 1,HL1210-3 can bind to human PD-L1 with high activity (EC₅₀=5.539 ng/ml).

Example 3: HL1210-3 Mouse mAb Blocked Human PD-L1 Binding to itsReceptor PD-1

Receptor Blocking Assay by Using Recombinant Human PD-L1

To evaluate the blocking effect of HL1210-3 mouse mAb on recombinanthuman PD-L1 to bind to its receptor PD-1, the ELISA based receptorblocking assay was employed. Briefly, microtiter plates were coated withhuman PD-L1-Fc protein at 1 μg/ml in PBS, 100 μl/well at 4° C.overnight, then blocked with 100 μl/well of 5% BSA. 50 μl biotin-labeledhuman PD-1-Fc protein and 3-fold dilutions of HL1210-3 antibodiesstarting from 2 μg/ml at 50 μl were added to each well and incubated for1 hour at 37° C. The plates were washed with PBS/Tween and thenincubated with Streptavidin-HRP for 1 hour at 37° C. After washing, theplates were developed with TMB substrate and analyzed byspectrophotometer at OD 450-630 nm. As shown in FIG. 2, HL1210-3 canefficiently inhibit the binding of human PD-L1 to human PD1 atIC₅₀=0.7835 nM.

Receptor Blocking Assay by Using Mammalian Cell Expressed Human PD-L1

To evaluate the blocking effect of HL1210-3 mouse mAb on human PD-L1expressed on mammalian cells to bind to its receptor PD-1, theFACS-based receptor blocking assay was used. Briefly, PDL1-CHOK1 cellswere firstly incubated with 3-fold serious diluted HL1210-3 mouse mAbstarting at 20 μg/ml at RT for 1 hour. After wash by FACS buffer (PBSwith 2% FBS), the biotin-labeled huPD-1 were added to each well andincubated at RT for 1 hour. Then, the Streptavidin-PE were added to eachwell for 0.5 hour post twice wash with FACS buffer. The meanfluorescence intensity (MFI) of PE were evaluated by FACSAriaIII. Asshown in FIG. 3, the HL1210-3 antibody can highly efficiently inhibitthe binding of PD-1 on PD-L1 expressed on mammalian cells at IC50 of2.56 nM with 92.6% top inhibition rate.

${\%\mspace{14mu}{of}\mspace{14mu}{inhibition}} = {\left( {1 - \frac{{MFI}\mspace{14mu}{of}\mspace{14mu}{testing}\mspace{14mu}{antibody}}{{MFI}\mspace{14mu}{of}\mspace{14mu}{vehicle}\mspace{14mu}{control}}} \right) \times 100\%}$

Example 4: HL1210-3 Mouse mAb Promoted Human T Cell Immune Response

To evaluate the effect of HL1210-3 mouse mAb, the response of human Tcells assessed in a mixed lymphocyte reaction setting. Human DCs weredifferentiated from CD14+ monocytes in the presence of GM-CSF and IL-4for 7 days. CD4+ T cells isolated from another donor were thenco-cultured with the DCs and serial dilutions of anti-PD-L1 blockingantibody. At day 5 post-inoculation, the culture supernatant was assayedfor IFNγ production. The results indicated that the HL1210-3 antibodiescan dose-dependently promote IFNγ production, suggesting anti-PD-L1antibody can promote human T cell response (FIG. 4).

Example 5: The Binding Affinity of HL1210-3 Mouse mAb

The binding of the HL1210-3 antibodies to recombinant PD-L1 protein(human PD-L1-his taq) was tested with BIACORE™ using a capture method.The HL1210-3 mouse mAb was captured using anti-mouse Fc antibody coatedon a CM5 chip. A series dilution of human PD-L1-his taq protein wasinjected over captured antibody for 3 mins at a flow rate of 25 μg/ml.The antigen was allowed to dissociate for 900 s. All the experiment werecarried out on a Biacore T200. Data analysis was carried out usingBiacore T200 evaluation software. The result are shown in FIG. 5 andTable 6 below.

TABLE 6 Binding Kinetics of HL1210-3 to recombinant human PD-L1 Antibodyka (1/Ms) kd (1/s) KD (M) HL1210-3 1.61E+05 4.69E−05 2.93E−10

Example 6: Humanization of the HL1210-3 Mouse mAb

The mAb HL1210-3 variable region genes were employed to create ahumanized MAb. In the first step of this process, the amino acidsequences of the VH and VK of MAb HL1210-3 were compared against theavailable database of human Ig gene sequences to find the overallbest-matching human germline Ig gene sequences. For the light chain, theclosest human match was the O18/Jk2 and KV1-39*01/KJ2*04 gene, and forthe heavy chain the closest human match was the VH3-21 gene. VH3-11,VH3-23, VH3-7*01 and VH3-48 genes were also selected due to their closematches.

Humanized variable domain sequences were then designed where the CDR1(SEQ ID NO.4), 2 (SEQ ID NO.5) and 3 (SEQ ID NO.6) of the HL1210-3 lightchain were grafted onto framework sequences of the O18/Jk2 andKV1-39*01/KJ2*04 gene, and the CDR1 (SEQ ID NO.1), 2 (SEQ ID NO.2), and3 (SEQ ID NO.3) sequences of the HL1210-3 VH were grafted onto frameworksequences of the VH3-21, VH3-11, VH3-23, VH3-48 or VH3-7*01 gene. A 3Dmodel was then generated to determine if there were any frameworkpositions where replacing the mouse amino acid to the human amino acidcould affect binding and/or CDR conformation. In the case of the lightchain, 22S, 43S, 60D, 63T and 42Q (Kabat numbering, see Table 7) inframework were identified. In the case of the heavy chain, 1E, 37V, 40T,44S, 49A, 77N, 91I, 94R and 108T in the framework was involved inback-mutations.

TABLE 7 Humanization Design Construct Mutation VH Design I: VH3-21/JH6Hu1210 VH Chimera Hu1210 VH.1 CDR-grafted Hu1210 VH.1a S49A Hu1210 VH.1bS49A, G44S, Y91I VH Design II: VH3-11/JH6 Hu1210 VH.2 CDR-grafted, Q1EHu1210 VH.2a Q1E, S49A Hu1210 VH.2b Q1E, I37V, S49A, G44S, Y91I VHDesign III: VH3-23/JH6 Hu1210 VH.3 CDR-grafted, K94R Hu1210 VH.3a G44S,S49A, Y91I, K94R VH Design IV: VH3-48/JH6 Hu1210 VH.4 CDR-grafted Hu1210VH.4a S49A Hu1210 VH.4b S49A, G44S, Y91I Hu1210 VH.4c D52E, S49A, G44S,Y91I Hu1210 VH.4d G53A, S49A, G44S, Y91I Hu1210 VH.4e G53V, S49A, G44S,Y91I VH Design V: VH3-7*01/HJ1*01 Hu1210 VH.5 CDR-grafted Hu1210 VH.5aH91I Hu1210 VH.5b H91I, H108T Hu1210 VH.5c H91I, H77N Hu1210 VH.5d H91I,H77N, H40T VK Design I: 018/Jk2 Hu1210 Vk Chimera Hu1210 Vk.1CDR-grafted Hu1210 Vk.1a A43S VK Design II: KV1-39*01/KJ2*04 Hu1210 Vk.2CDR-grafted Hu1210 Vk.2a L60D, L63T Hu1210 Vk.2b L60D, L63T, L42Q, L43SHu1210 Vk.2c L60D, L63T, L42Q, L43S, T22S

The amino acid and nucleotide sequences of some of the humanizedantibody are listed in Table 8 below.

TABLE 8  Humanized antibody sequences (bold indicates CDR) SEQ ID NameSequence NO: HL1210-VHEVKLVESGGDLVKPGGSLKLSCAASGFTFSSYDMSWVRQTPEKSLEWVAT 7ISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMSSLRSEDTALYICAREF GKRYALDYWGQGTSVTVSSHu1210 VH.1 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST 8ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF GKRYALDYWGQGTIVIVSS Hu1210 VH.1a EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 9ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF GKRYALDYWGQGTIVIVSS Hu1210 VH.1b EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 10ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTIVIVSS Hu1210 VH.2 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWIRQAPGKGLEWVST 11ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.2a EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWIRQAPGKGLEWVAT 12ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.2b EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 13ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.3 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST 14ISDGGGYIYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.3a EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 15ISDGGGYIYYSDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.4 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVST 16ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.4a EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 17ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.4b EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 18ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.4c EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 19ISEGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.4d EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 20ISDAGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.4e EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKSLEWVAT 21ISDVGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYICAREF GKRYALDYWGQGTTVTVSS Hu1210 VH.5 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 22ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAREF GKRYALDYWGQGTLVTVSS HU1210 VH.5a EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 23ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTLVTVSS HU1210 VH.5b EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 24ISDGGGYIYYSDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTTVTVSS HU1210 VH.5C EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQAPGKGLEWVAT 25ISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTLVTVSS HU1210 VH.5d EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDMSWVRQTPEKSLEWVAT 26ISDGGGYIYYSDSVKGRFTISRDNAKNNLYLQMNSLRAEDTAVYICAREF GKRYALDYWGQGTLVTVSS HL1210-VK DIVMTQSHKFMSTSVGDRVSISCKASQDVTPAVAWYQQKPGQSPKLLIYS  27TSSRYTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYYCQQHYTTPLTFGA GTKLELK Hu1210 VK.1DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS  28TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK Hu1210 VK.1aDIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKSPKLLIYS  29TSSRYTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQHYTTPLTFGQ GTKLEIK Hu1210 Vk.2DIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS  30TSSRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR Hu1210 Vk.2aDIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGKAPKLLIYS  31TSSRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR Hu1210 Vk.2bDIQMTQSPSSLSASVGDRVTITCKASQDVTPAVAWYQQKPGQSPKLLIYS  32TSSRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR Hu1210 Vk.2cDIQMTQSPSSLSASVGDRVTISCKASQDVTPAVAWYQQKPGQSPKLLIYS  33TSSRYTGVPDRFTGSGSGTDFTLTISSLQPEDFATYYCQQHYTTPLTFGQ GTKLEIKR HL1210 VHGAGGTGAAGCTGGTGGAGAGCGGCGGAGATCTGGTGAAGCCTGGCGGCAGCCTGAAGCTG 34AGCTGTGCCGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGGCAGACCCCCGAGAAGAGCCTGGAGTGGGTGGCCACCATCAGCGATGGCGGCGGCTACATCTACTACAGCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAACCTGTACCTGCAGATGAGCAGCCTGAGGAGCGAGGACACCGCCCTGTACATCTGCGCCAGGGAGTTCGGCAAGAGGTACGCCCTGGACTACTGGGGACAGGGCACCAGCGTGACCGTGAGCAGC Hu1210 VH.1GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG 35AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.1aGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG 36AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.1bGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG 37AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.2GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG 38AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.2aGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG 39AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGATCAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.2bGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGAAGCCCGGAGGCAGCCTGAGACTG 40AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.3GAGGTGCAGCTGCTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 41AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.3aGAGGTGCAGCTGCTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 42AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.4GAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 43AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGAGCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.4aGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 44AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAGGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACTACTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.4bGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 45AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.4cGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 46AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGAAGGCGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210_VH.4dGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 47AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGCGGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210_VH.4eGAGGTGCAGCTGGTGGAGAGCGGAGGAGGACTGGTGCAACCCGGAGGCAGCCTGAGACTG 48AGCTGCGCTGCCAGCGGCTTCACCTTCAGCAGCTACGACATGAGCTGGGTGAGACAGGCCCCTGGCAAAAGCCTGGAGTGGGTGGCCACCATCTCCGATGTTGGCGGCTACATCTATTACTCCGACAGCGTGAAGGGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACAGCCTGTACCTGCAGATGAACAGCCTGAGGGATGAGGACACCGCCGTGTACATCTGCGCCAGGGAGTTCGGCAAAAGGTACGCCCTGGACTACTGGGGCCAGGGCACAACCGTGACCGTGAGCAGC Hu1210 VH.5GAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG 49TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATTACTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210 VH.5aGAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG 50TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210 VH.SbGAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG 51TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGTTCACCATCTCCCGGGACAACGCCAAGAACAACCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210 VH.ScGAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG 52TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGACCCCTGAGAAGAGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGITCACCATCTCCCGGGACAACGCCAAGAACAACCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACACTGGTGACAGTGAGCTCC Hu1210_VH.5dGAGGTGCAGCTGGTGGAGTCCGGAGGAGGCCTGGTGCAACCTGGAGGCTCCCTGAGGCTG 53TCCTGTGCCGCTTCCGGCTTCACCTTCAGCTCCTACGATATGAGCTGGGTGAGGCAGGCTCCTGGAAAGGGCCTGGAGTGGGTGGCCACCATCTCCGACGGAGGCGGCTACATCTACTACTCCGACTCCGTGAAGGGCAGGITCACCATCTCCCGGGACAACGCCAAGAACTCCCTGTACCTGCAGATGAACTCTCTCAGGGCTGAGGACACCGCCGTGTATATCTGCGCCAGGGAGTTTGGCAAGAGGTACGCCCTGGATTACTGGGGCCAGGGCACAACCGTGACAGTGAGCTCC HL1210 VKGACATCGTGATGACCCAGAGCCACAAGTTCATGAGCACCAGCGTGGGCGATAGGGTGAGC 54ATCAGCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCGACAGGTTCACAGGAAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCGTGCAGGCCGAGGACCTGGCCGTGTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCGCCGGCACCAAGCTGGAGCTGAAG Hu1210 VK.1GACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACC 55ATCACCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCAGCAGGTTTAGCGGAAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG Hu1210 VK.1aGACATCCAGATGACCCAGAGCCCTAGCAGCCTGAGCGCTAGCGTGGGCGACAGGGTGACC 56ATCACCTGCAAGGCCAGCCAGGATGTGACCCCTGCCGTGGCCTGGTACCAGCAGAAGCCCGGCAAGTCCCCCAAGCTGCTGATCTACAGCACCAGCAGCAGGTACACCGGCGTGCCCAGCAGGTTTAGCGGAAGCGGCAGCGGCACCGACTTCACCTTCACCATCAGCAGCCTGCAGCCCGAGGACATCGCCACCTACTACTGCCAGCAGCACTACACCACCCCTCTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAG Hu1210 VK.2GACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC 57ATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGIGGCCTGGTATCAACAGAAGCCTGGCAAGGCTCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCTCCAGGTTTAGCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGG Hu1210 VK.2aGACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC 58ATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGIGGCCTGGTATCAACAGAAGCCTGGCAAGGCTCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGACAGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGG Hu1210 VK.2bGACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC 59ATCACCTGCAAGGCCAGCCAGGACGTGACACCTGCTGIGGCCTGGTATCAACAGAAGCCTGGCCAGAGCCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGACAGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGG Hu1210 VK.2cGACATTCAGATGACCCAGTCCCCTAGCAGCCTGTCCGCTTCCGTGGGCGACAGGGTGACC 60ATCAGCTGCAAGGCCAGCCAGGACGTGACACCTGCTGIGGCCTGGTATCAACAGAAGCCTGGCCAGAGCCCTAAGCTCCTGATCTACAGCACATCCTCCCGGTACACCGGAGTGCCCGACAGGTTTACCGGCAGCGGCTCCGGCACCGATTTCACCCTGACCATTTCCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACACCCCTGACCTTCGGCCAGGGCACCAAGCTGGAGATCAAGCGG

The humanized VH and VK genes were produced synthetically and thenrespectively cloned into vectors containing the human gamma 1 and humankappa constant domains. The pairing of the human VH and the human VKcreated the 40 humanized antibodies (see Table 9).

TABLE 9 Humanized antibodies with their VH an VL regions Hu1210 Hu1210Hu1210 Hu1210 Hu1210 Hu1210 Hu1210 VH Vk VH.1 VH.1a VH.1b VH.2 VH.2aVH2.b VH Hu1210 Vk.1 Hu1210-1 Hu1210-2 Hu1210-3 Hu1210-4 Hu1210-5 Hu1210Vk.1a Hu1210-7 Hu1210-8 Hu1210-9 Hu1210-10 Hu1210-11 Hu1210 Vk H1210chimera Hu1210 Hu1210 Hu1210 Hu1210 Hu1210 VH Vk VH.3 VH.3a VH.4 VH.4aVH.4b Hu1210 Vk.1 Hu1210-13 Hu1210-14 Hu1210-15 Hu1210-16 Hu1210-17Hu1210 Vk.1a Hu1210-18 Hu1210-19 Hu1210-20 Hu1210-21 Hu1210-22 Hu1210HU1210 HU1210 HU1210 HU1210 VH VK VH.5 VH.5a VH.5b VH.5c VH.5d Hu1210Vk.2 Hu1210-23 Hu1210-27 Hu1210-31 Hu1210-32 Hu1210-36 Hu1210 Vk.2aHu1210-24 Hu1210-28 Hu1210-33 Hu1210-37 Hu1210 Vk.2b Hu1210-25 Hu1210-29Hu1210-34 Hu1210-38 Hu1210 Vk.2c Hu1210-26 Hu1210-30 Hu1210-35 Hu1210-39Hu1210 Hu1210 Hu1210 VH Vk VH.4c VH.4d VH.4e Hu1210 Vk.1 Hu1210-40Hu1210-41 Hu1210-42

Example 7: The Antigen Binding Properties of Humanized PD-L1 Antibodies

Binding Property to Recombinant Human PD-L1

To evaluate the antigen binding activity, the humanized antibodies weresubjected to ELISA test. Briefly, microtiter plates were coated withhuman PD-L1-Fc protein at 0.1 μg/ml in PBS, 100 μl/well at 4° C.overnight, then blocked with 100 μl/well of 5% BSA. Five-fold dilutionsof humanized antibodies starting from 10 μg/ml were added to each welland incubated for 1-2 hours at RT. The plates were washed with PBS/Tweenand then incubate with goat-anti-mouse IgG antibody conjugated withHorse Radish Peroxidase (HRP) for 1 hour at RT. After washing, theplates were developed with TMB substrate and analyzed byspectrophotometer at OD 450-630 nm. As shown in FIG. 6, all thehumanized antibodies show comparable binding efficacy to human PD-L1 incontact to chimeric antibody.

Binding Property to Mammalian Expressed Human PD-L1

To evaluate the antigen binding property, the humanized antibodies wereanalyzed for its binding to mammalian expressed PD-L1 by FACS. Briefly,PDL1-CHOK1 cells were firstly incubated with 5-fold serious dilutedhumanized antibodies starting at 2 μg/ml at RT for 1 hour. After wash byFACS buffer (PBS with 2% FBS), the alexa 488-anti-human IgG antibody wasadded to each well and incubated at RT for 1 hour. The MFI of Alexa 488were evaluated by FACSAriaIII. As shown in the FIG. 7, all the humanizedantibodies can high efficiently bind to PD-L1 expressed on mammaliancells, which was comparable with chimeric antibody.

To explore the binding kinetics of the humanized antibody, this exampleperformed the affinity ranking by using Octet Red 96. As shown in Table10, hu1210-3, hu1210-8, hu1210-9, hu1210-14, hu1210-17, hu1210-1 andHu1210-22 show better affinity, which is comparable with chimericantibody.

TABLE 10 Affinity ranking of humanized antibodies Antibody KD (M)kon(1/Ms) kdis(1/s) Hu1210 (mIgG) 7.16E−09 3.94E+05 2.83E−03 H1210chimera 1.07E−09 1.62E+05 1.73E−04 Hu1210-1 4.25E−09 7.10E+04 3.02E−04Hu1210-2 3.23E−09 7.78E+04 2.51E−04 Hu1210-3 2.64E−09 8.62E+04 2.28E−04Hu1210-4 7.68E−09 7.12E+04 5.46E−04 Hu1210-5 4.83E−09 7.93E+04 3.83E−04Hu1210-7 4.78E−09 8.45E+04 4.04E−04 Hu1210-8 1.64E−09 7.72E+04 1.27E−04Hu1210-9 2.33E−09 8.37E+04 1.95E−04 Hu1210-10 7.03E−09 8.59E+04 6.04E−04Hu1210-11 4.18E−09 7.54E+04 3.15E−04 Hu1210-13 4.36E−09 8.38E+043.66E−04 Hu1210-14 2.34E−09 8.41E+04 1.97E−04 Hu1210-15 4.45E−097.87E+04 3.50E−04 Hu1210-16 3.14E−09 8.41E+04 2.64E−04 Hu1210-172.20E−09 8.17E+04 1.80E−04 Hu1210-18 4.50E−09 7.92E+04 3.57E−04Hu1210-19 2.50E−09 9.03E+04 2.25E−04 Hu1210-20 4.51E−09 8.87E+044.00E−04 Hu1210-21 3.12E−09 9.39E+04 2.93E−04 Hu1210-22 2.56E−099.00E+04 2.30E−04Full Kinetic Affinity of Humanized Antibodies by Biacore®

The binding of the humanized antibodies to recombinant PD-L1 protein(human PD-L1-his taq) was tested by BIACORE™ using a capture method. TheHL1210-3 mouse mAb were captured using anti-mouse Fc antibody coated ona CM5 chip. A series dilution of human PD-L1-his taq protein wasinjected over captured antibody for 3 mins at a flow rate of 25 μg/ml.The antigen was allowed to dissociate for 900 s. All the experiment werecarried out on a Biacore T200. Data analysis was carried out usingBiacore T200 evaluation software and is shown in Table 11 below.

TABLE 11 Affinity by Biacore Antibody ka (1/Ms) kd (1/s) KD (M) Hu1210-89.346E+4 7.169E−5 7.671E−10 Hu1210-9 9.856E+4 4.528E−5 4.594E−10Hu1210-14 1.216E+5 5.293E−5 4.352E−10 Hu1210-16 9.978E+4 6.704E−56.720E−10 Hu1210-17 1.101E+5 2.128E−5 1.933E−10 Hu1210-28 1.289E+51.080E−4 8.378E−10 Hu1210-31 1.486E+5 1.168E−4 7.862E−10 Hu1210-361.461E+5 7.852E−5 5.376E−10 Hu1210-40  8.77E+04  1.31E−04  1.49E−09Hu1210-41  9.17E+04  3.46E−05  3.78E−10 Hu1210-42  8.68E+04  7.53E−05 8.67E−10 1210 Chimera 1.236E+5 3.265E−5 2.642E−10Cross Species Activity

To evaluate the binding of humanized antibodies to huPD-L1, Mouse PD-L1,Rat PD-L1, Rhesus PD-L1, the antibodies were performed for the ELISAtesting. Briefly, microtiter plates were coated with human, mouse, ratand rhesus PD-L1-Fc protein at 1 μg/ml in PBS, 100 μl/well at 4° C.overnight, then blocked with 100 μl/well of 5% BSA. Three-fold dilutionsof humanized antibodies starting from 1 μg/ml were added to each welland incubated for 1-2 hours at RT. The plates were washed with PBS/Tweenand then incubate with goat-anti-mouse IgG antibody conjugated withHorse Radish Peroxidase (HRP) for 1 hour at RT. After washing, theplates were developed with TMB substrate and analyzed byspectrophotometer at OD 450-630 nm. The Hu1210-41 antibody can bind torhesus PD-L1 with lower affinity and cannot bind to rat and mouse PD-L1(FIG. 8).

Human Rhesus Rat Mouse EC50 0.215 nM 0.628 nM No binding No bindingFamily Member Specificity

To evaluate the binding of humanized anti-PD-L1 antibody to human B7family and other immune checkpoint, the antibody was evaluate for itsbinding to B7-H1 (PD-L1), B7-DC, B7-1, B7-2, B7-H2, PD-1, CD28, CTLA4,ICOS and BTLA by ELISA. As shown in FIG. 9, the Hu1210-41 antibody canonly specifically binding to B7-H1 (PD-L1).

Example 8: Humanized Antibodies Blocked Activity of Human PD-L1 to PD-1

Cell Based Receptor Blocking Assay

To evaluate the blocking effect of humanized antibodies on human PD-L1expressed on mammalian cells to bind to its receptor PD-1, theFACS-based receptor blocking assay was employed. Briefly, PDL1-CHOK1cells were firstly incubated with 3-fold serious diluted HL1210-3 mousemAb starting at 20 μg/ml at RT for 1 hour. After wash by FACS buffer(PBS with 2% FBS), the biotin-labeled huPD-1 were added to each well andincubated at RT for 1 hour. Then, the Streptavidin-PE were added to eachwell for 0.5 hour post twice wash with FACS buffer. The meanfluorescence intensity (MFI) of PE were evaluated by FACSAriaIII.

${\%\mspace{14mu}{of}\mspace{14mu}{inhibition}} = {\left( {1 - \frac{{MFI}\mspace{14mu}{of}\mspace{14mu}{testing}\mspace{14mu}{antibody}}{{MFI}\mspace{14mu}{of}\mspace{14mu}{vehicle}\mspace{14mu}{control}}} \right) \times 100\%}$

As shown in Table 12 below, Hu1210-3, Hu1210-9, Hu1210-8, Hu1210-14,Hu1210-17, Hu1210-19 and Hu1210-22 antibodies show comparable efficacywith chimeric antibody to blocking the binding of PD-L1 to PD-1.

TABLE 12 PD-1 receptor blocking assay Bio-PD1(30 μg/ml) TOP EC50 H1210chimera 87.16 3.961 Hu1210-8 86.35 4.194 Hu1210-9 85.7 4.038 Hu1210-1688.02 5.436 Hu1210-17 80.88 4.424 Hu1210-3 84.28 3.693 Hu1210-14 79.563.572 Hu1210-19 87.45 4.52 Hu1210-22 85.83 4.505 Hu1210-27 103.9 11.48Hu1210-31 92.91 6.179 Hu1210-36 91.75 8.175Receptor Blocking Assay by Using Recombinant Human PD-L1

There are two receptors i.e. PD-1 and B7-1 for human PD-L1. To explorethe blocking property of humanized PD-L1 antibody to these two proteins,the protein based receptor blocking assay was employed here. Briefly,microtiter plates were coated with human PD-L1-Fc protein at 1 μg/ml inPBS, 100 μl/well at 4° C. overnight, then blocked with 200 μl/well of 5%BSA at 37° C. for 2 hr. 50 μl biotin-labeled human PD-1-Fc orB7-1vprotein and 5-fold dilutions of PD-L1 antibodies starting from 100nM at 50 μl were added to each well and incubated for 1 hour at 37° C.The plates were washed with PBS/Tween and then incubate withStreptavidin-HRP for 1 hour at 37° C. After washing, the plates weredeveloped with TMB substrate and analyzed by spectrophotometer at OD 450nm. As shown in FIGS. 10 and 11, Hu1210-41 can efficiently inhibit thebinding of human PD-L1 to human PD1 and B7-1.

Example 9: Humanized Antibody Promoted Human T Cell Immune Response

Mixed Lymphocyte Reaction Assay

To evaluate the in vitro function of humanized antibodies, the responseof human T cells assessed in a mixed lymphocyte reaction setting. HumanDCs were differentiated from CD14+ monocytes in the presence of GM-CSFand IL-4 for 7 days. CD4+ T cells isolated from another donor were thenco-cultured with the DCs and serial dilutions of anti-PD-L1 blockingantibody. At day 5 post-inoculation, the culture supernatant was assayedfor IL-2 and IFNγ production. The results indicated that the Hu1210-8,Hu1210-9, Hu1210-16 and Hu1210-17 antibodies can dose-dependentlypromote IL-2 and IFNγ production, suggesting anti-PD-L1 antibodies canpromote human T cell response.

CMV Recall Assay

To evaluate the in vitro function of humanized antibodies, the responseof human T cells assessed in CMV recall assay. Human PBMCs werestimulated with 1 μg/ml CMV antigen in the presence of serious dilutedhumanized antibodies. As shown in FIGS. 12 and 13 the Hu1210-40,Hu1210-41 and Hu1210-17 can dose dependently promote the IFNγproduction.

Example 10: Tumor Growth Inhibition by Anti-PD-L1 mAb

Cells from the human lung adenocarcinoma cell line HCC827 will begrafted into NOD scid gamma (NSG) mice. NSG mice are NOD scid gammadeficient and the most immunodeficient mice making them ideal recipientsfor human tumor cell and PBMC grafting. 10 days post-graft, human PBMCswill be transplanted into the tumor-bearing mice. Approximately 20 dayspost-graft, once the tumor volume has reached 100-150 mm³, PD-L1antibody will be administered to the mice every other day at 5 mg/kg.Tumor volume will be monitored every other day in conjunction withantibody administration. As shown in FIG. 14, Hu1210-31 can inhibit thetumor growth by 30% at 5 mg/kg. Hu1210-41 antibody can dose-dependentlyinhibit the tumor growth, while the tumor weight was alsodose-dependently suppressed by Hu1210-41 antibody (FIG. 15).

Example 11: Computer Simulation of Further Variation and Optimization ofthe Humanized Antibodies

It was contemplated that certain amino acid residues within the CDRregions or the framework regions could be changed to further improve orretain the activity and/or stability of the antibodies. Variants weretested, with a computational tool (VectorNTI, available atwww.ebi.ac.uk/tools/msa/clustalo/), with respect to their structural,conformational and functional properties, and those (within the CDRregions) that showed promises are listed in the tables blow.

TABLE 13  VH and VL CDRs and their variants suitable forinclusion in humanized antibodies Name Sequence SEQ ID NO: VH CDR1 SY DMS   1 TYDMS 61 CYDMS 62 SFDMS 63 SHDMS 64 SWDMS 65 SYDMT 66 SYDMC 67VH CDR2 TISDGG GY IYYSD SV KG 2 TISDGGAYIYYSDSVKG 68 TISDGGPYIYYSDSVKG69 TISDGGGFIYYSDSVKG 70 TISDGGGHIYYSDSVKG 71 TISDGGGWIYYSDSVKG 72TISDGGGYIYYSDTVKG 73 TISDGGGYIYYSDCVKG 74 TISDGGGYIYYSDSLKG 75TISDGGGYIYYSDSIKG 76 TISDGGGYIYYSDSMKG 77 VH CDR3 EFGKRY ALDY  3QFGKRYALDY 78 DFGKRYALDY 79 NFGKRYALDY 80 EYGKRYALDY 81 EHGKRYALDY 82EWGKRYALDY 83 EFAKRYALDY 84 EFPKRYALDY 85 EFGRRYALDY 86 EFGKKYALDY 87EFGKRFALDY 88 EFGKRHALDY 89 EFGKRWALDY 90 VL CDR1 KA S QDVTPAVA 4KATQDVTPAVA 91 KACQDVTPAVA 92 VL CDR2 STSSRY T 5 TTSSRYT 93 CTSSRYT 94SSSSRYT 95 SMSSRYT 96 SVSSRYT 97 STTSRYT 98 STCSRYT 99 STSTRYT 100STSCRYT 101 STSSKYT 102 STSSRFT 103 STSSRHT 104 STSSRWT 105 VL CDR3 QQHYTTPLT 6 EQHYTTPLT 106 DQHYTTPLT 107 NQHYTTPLT 108 QEHYTTPLT 109QDHYTTPLT 110 QNHYTTPLT 111 Underline: hotspot mutation residues andtheir substitutes

Example 12: Identification of PD-L1 Epitope

This study was conducted to identify amino acid residues involved in thebinding of PD-L1 to the antibodies of the present disclosure.

An alanine-scan library of PD-L1 was constructed. Briefly, 217 mutantclones of PD-L1 were generated on Integral Molecular's proteinengineering platform. Binding of Hu1210-41 Fab to each variant in thePD-L1 mutation library was determined, in duplicate, by high-throughputflow cytometry. Each raw data point had background fluorescencesubtracted and was normalized to reactivity with PD-L1 wild-type (WT).For each PD-L1 variant, the mean binding value was plotted as a functionof expression (control anti-PD-L1 mAb reactivity). To identifypreliminary critical clones (circles with crosses), thresholds (dashedlines) of >70% WT binding to control MAb and <30% WT reactivity toHu1210-41 Fab were applied (FIG. 16). Y134, K162, and N183 of PDL1 wereidentified as required residues for Hu1210-41 binding. The lowreactivity of N183A clone with Hu1210-41 Fab suggests that it is themajor energetic contributor to Hu1210-41 binding, with lessercontributions by Y134 and K162.

The critical residues (spheres) were identified on a 3D PD-L1 structure(PDB ID #5JDR, Zhang et al., 2017), illustrated in FIG. 17. Theseresidues, Y134, K162, and N183, therefore, constitute an epitope ofPD-L1 responsible for binding to antibodies of various embodiments ofthe present disclosure.

It is interesting to note that Y134, K162, and N183 are all locatedwithin the IgC domain of the PD-L1 protein. Both PD-1 and PD-L1'sextracellular portions have an IgV domain and an IgC domain. It iscommonly known that PD-L1 binds to PD-1 through bindings between theirIgV domains. Unlike such conventional antibodies, however, Hu1210-41binds to the IgC domain, which would have been expected to beineffective in inhibiting PD-1/PD-L1 binding. This different epitope ofHu1210-41, surprisingly, likely contributes to the excellent activitiesof Hu1210-41.

The present disclosure is not to be limited in scope by the specificembodiments described which are intended as single illustrations ofindividual aspects of the disclosure, and any compositions or methodswhich are functionally equivalent are within the scope of thisdisclosure. It will be apparent to those skilled in the art that variousmodifications and variations can be made in the methods and compositionsof the present disclosure without departing from the spirit or scope ofthe disclosure. Thus, it is intended that the present disclosure coverthe modifications and variations of this disclosure provided they comewithin the scope of the appended claims and their equivalents.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference

What is claimed is:
 1. A method of treating cancer in a patient in needthereof, comprising administering to the patient an effective amount ofan antibody or fragment thereof, wherein the antibody or fragmentthereof has specificity to a human Programmed death-ligand 1 (PD-L1)protein and comprises a VH CDR1 comprising the amino acid sequence ofSEQ ID NO: 1, a VH CDR2 comprising the amino acid sequence of SEQ ID NO:2, a VH CDR3 comprising the amino acid sequence of SEQ ID NO: 3, a VLCDR1 comprising the amino acid sequence of SEQ ID NO: 4, a VL CDR2comprising the amino acid sequence of SEQ ID NO: 5, and a VL CDR3comprising the amino acid sequence of SEQ ID NO:
 6. 2. The method ofclaim 1, wherein the cancer is selected from the group consisting ofbladder cancer, liver cancer, colon cancer, rectal cancer, endometrialcancer, leukemia, lymphoma, pancreatic cancer, small cell lung cancer,non-small cell lung cancer, breast cancer, urethral cancer, head andneck cancer, gastrointestinal cancer, stomach cancer, oesophagealcancer, ovarian cancer, renal cancer, melanoma, prostate cancer andthyroid cancer.
 3. A method of treating cancer in a patient in needthereof, comprising: (a) treating a T cell, in vitro, with an antibodyor fragment thereof; and (b) administering the treated T cell to thepatient, wherein the antibody or fragment thereof has specificity to ahuman Programmed death-ligand 1 (PD-L1) protein and comprises a VH CDR1comprising the amino acid sequence of SEQ ID NO: 1, a VH CDR2 comprisingthe amino acid sequence of SEQ ID NO: 2, a VH CDR3 comprising the aminoacid sequence of SEQ ID NO: 3, a VL CDR1 comprising the amino acidsequence of SEQ ID NO: 4, a VL CDR2 comprising the amino acid sequenceof SEQ ID NO: 5, and a VL CDR3 comprising the amino acid sequence of SEQID NO:
 6. 4. The method of claim 3, further comprising, prior to step(a), isolating the cell from an individual.
 5. The method of claim 4,wherein the cell is isolated from the patient.
 6. The method of claim 5,wherein the cell is isolated from a donor individual different from thepatient.
 7. The method of claim 3, wherein the T cell is atumor-infiltrating T lymphocyte, a CD4+ T cell, or a CD8+ T cell.
 8. Themethod of claim 1, wherein the antibody or fragment therefor furthercomprises a heavy chain constant region, a light chain constant region,an Fc region, or the combination thereof.
 9. The method of claim 1,wherein the antibody or fragment thereof is humanized.
 10. A method oftreating cancer in a patient in need thereof, comprising administeringto the patient an effective amount of an antibody or fragment thereof,wherein the antibody or fragment thereof has specificity to a humanProgrammed death-ligand 1 (PD-L1) protein and comprises a heavy chainvariable region comprising an amino acid sequence selected from thegroup consisting of SEQ ID NO: 7-26 and a light chain variable regioncomprising an amino acid sequence selected from the group consisting ofSEQ ID NO: 27-33.
 11. The method of claim 10, wherein the heavy chainvariable region comprises the amino acid sequence of SEQ ID NO: 20 andthe light chain variable region comprises the amino acid sequence of SEQID NO: 28.